FLT3L-based chimeric proteins

ABSTRACT

The present invention relates, inter alia, to compositions and methods, including chimeric proteins comprising an extracellular domain of FMS like tyrosine kinase 3 ligand (FLT3L) and an extracellular domain of a Type II transmembrane protein that find use in the treatment of disease, such as cancer.

PRIORITY

This application is a continuation of International Application SerialNo. PCT/US2019/048922, filed Aug. 29, 2019, which claims the benefit of,and priority to, U.S. Provisional Application No. 62/724,596, filed Aug.29, 2018, the content of which is herein incorporated by reference inits entirety.

TECHNICAL FIELD

The present invention relates to, inter alia, compositions and methods,including chimeric proteins that find use in the treatment of disease,such as immunotherapies for cancer and autoimmunity.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“SHK-010PC_SequenceListing_ST25”. The sequence listing is 40,839 bytesin size, and was created on Aug. 28, 2019. The sequence listing ishereby incorporated by reference in its entirety.

BACKGROUND

The immune system is central to the body's response to foreign entitiesthat can cause disease and to the body's response to cancer cells.However, many anti-cancer therapeutics do not directly stimulate and/oractivate the immune response. Thus, there remains a need to developtherapeutics that, at least, directly stimulate and/or activate apatient's anti-cancer immune response.

SUMMARY

Accordingly, in various aspects, the present invention provides forcompositions and methods that are useful for cancer immunotherapy. Forinstance, the present invention, in part, relates to specific chimericproteins that provide immune activating or co-stimulatory signals, e.g.,to expand and activate dendritic cells.

The present invention relates to chimeric proteins comprising anextracellular domain of FMS like tyrosine kinase 3 ligand (FLT3L) and anextracellular domain of a Type II transmembrane protein. In embodiments,such chimeric proteins have “dual costimulatory” capability, since eachdomain of the chimeric protein can independently stimulate a singleimmune system cell or can simultaneously or contemporaneously stimulatea pair of immune system cells.

The extracellular domain of a Type I transmembrane protein, includingFLT3L, is located at the protein's amino terminus see, by way ofnon-limiting example, FIG. 1A, left protein), whereas the extracellulardomain of a Type II transmembrane protein is located at the protein'scarboxy terminus (see, by way of non-limiting example, FIG. 1A, rightprotein). The extracellular domain of Type I transmembrane protein,including FLT3L, contains the functional domains that are responsiblefor interacting with other binding partners (either ligands orreceptors) in the extracellular environment (see, FIG. 1B, left protein)and the extracellular domain of Type II transmembrane protein containsthe functional domains that are responsible for interacting with otherbinding partners (either ligands or receptors) in the extracellularenvironment (see, FIG. 1B, right protein).

Aspects of the present invention provide a chimeric protein comprising ageneral structure of: N terminus-(a)-(b)-(c)-C terminus, where (a) is afirst domain comprising an extracellular domain of FMS like tyrosinekinase 3 ligand (FLT3L), (b) is a linker adjoining the first domain andthe second domain, e.g., the linker comprising at least one cysteineresidue capable of forming a disulfide bond and/or comprising ahinge-CH2-CH3 Fc domain, and (c) is a second domain comprising anextracellular domain of a Type II transmembrane protein; wherein thelinker connects the first domain and the second domain. See, by way ofnon-limiting examples, FIG. 1C and FIG. 1D.

Aspects of the present invention provide a chimeric protein comprising ageneral structure of: N terminus-(a)-(b)-(c)-C terminus, where (a) is afirst domain comprising an extracellular domain of FMS like tyrosinekinase 3 ligand (FLT3L), (b) is a linker adjoining the first domain andthe second domain, e.g., the linker comprising at least one cysteineresidue capable of forming a disulfide bond and/or comprising ahinge-CH2-CH3 Fc domain, and (c) is a second domain comprising anextracellular domain of one of CD40L, OX40L, 4-1BBL, LIGHT, CD30L,TRAIL, FasL, APRIL, BAFF, TWEAK, TL1A, CD70, and GITRL. A chimericprotein of these aspects may have the structure of shown in FIG. 1C orFIG. 1D.

Other aspects of the present invention provide an expression vectorcomprising a nucleic acid which encodes a chimeric protein as disclosedherein.

Yet other aspects of the present invention provide a host cellcomprising the expression vector disclosed herein.

In aspects, the present invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of the chimeric protein asdisclosed herein.

In other aspects, the present invention provides a method of treatingcancer or treating an inflammatory disorder due to viral infection. Themethod comprising a step of administering to a subject in need thereofan effective amount of a pharmaceutical composition as disclosed herein.

In yet other aspects, the present invention provides a method ofmodulating a patient's immune response. The method comprising a step ofadministering to a subject in need thereof an effective amount of apharmaceutical composition as disclosed herein.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1D show schematic illustrations of Type I transmembraneproteins (FIG. 1A and FIG. 1B, left proteins) and Type II transmembraneproteins (FIG. 1A and FIG. 1B, right proteins). A Type I transmembraneprotein and a Type II transmembrane protein may be engineered such thattheir transmembrane and intracellular domains are omitted and thetransmembrane proteins' extracellular domains are adjoined using alinker sequence to generate a single chimeric protein. As shown in FIG.1C and FIG. 1D, the extracellular domain of a Type I transmembraneprotein, e.g., FLT3L, and the extracellular domain of a Type IItransmembrane protein are combined into a single chimeric protein. FIG.1C depicts the linkage of the Type I transmembrane protein and the TypeII transmembrane protein by omission of the transmembrane andintracellular domains of each protein, and where the liberatedextracellular domains from each protein have been adjoined by a linkersequence. The extracellular domains in this depiction may include theentire amino acid sequence of the Type I protein (e.g., FLT3L) and/orType II protein which is typically localized outside the cell membrane,or any portion thereof which retains binding to the intended receptor orligand. Moreover, the chimeric protein comprises sufficient overallflexibility and/or physical distance between domains such that a firstextracellular domain (shown at the left end of the chimeric protein inFIG. 1C and FIG. 1D) is sterically capable of binding itsreceptor/ligand and/or a second extracellular domain (shown at the rightend of the chimeric protein in FIG. 1C and FIG. 1D) is stericallycapable of binding its receptor/ligand. FIG. 1D depicts adjoinedextracellular domains in a linear chimeric protein wherein eachextracellular ndomain of the chimeric protein is facing “outward”.

FIG. 2 shows immune inhibitory and immune stimulatory signaling proteinsand interactions that are relevant to the present invention (fromMahoney, Nature Reviews Drug Discovery 2015:14; 561-585).

FIG. 3A shows characterization of a murine FLT3L-Fc-4-1BBL chimericprotein by Western blot demonstrating the chimeric proteins native stateand tendency to form a multimer. Untreated samples (i.e., withoutreducing agent or deglycosylation agent) of the FLT3L-Fc-4-1BBL chimericprotein, e.g., control, were loaded into lane 2 in all the blots.Samples in lane 3 were treated with the reducing agent, βercaptoethanol.Samples in lane 4 were treated with a deglycosylation agent and thereducing agent. Each individual domain of the chimeric protein wasprobed using aanti-FLT3L, anti-Fc, or anti-4-1BBL antibody,respectively. FIG. 3B to FIG. 3D show ELISA data demonstrating thebinding affinity of mFLT3L domain of mFLT3L-Fc-4-1BBL (FIG. 3B), of themFc domain (FIG. 3C), and of the 4-1BBL domain (FIG. 3D) for theirrespective binding partners.

FIG. 4A shows characterization of a murine FLT3L-Fc-OX40L chimericprotein by Western blot demonstrating the chimeric proteins native stateand tendency to form a multimer. Untreated samples (i.e., withoutreducing agent or deglycosylation agent) of the FLT3L-Fc-CD40L chimericprotein, e.g., control, were loaded into lane 2 in all the blots.Samples in lane 3 were treated with the reducing agent,β-mercaptoethanol. Samples in lane 4 were treated with a deglycosylationagent and the reducing agent. Each individual domain of the chimericprotein was probed using an anti-FLT3L, anti-Fc, or anti-CD40L antibody,respectively. FIG. 4B to FIG. 4D show ELISA data demonstrating thebinding affinity of mFLT3L domain of mFLT3L-Fc-CD40L (FIG. 4B), the ofmFc domain (FIG. 4C), and of the mCD40L domain (FIG. 4D) for theirrespective binding partners.

FIG. 5A shows characterization of a murine FLT3L-Fc-OX40L chimericprotein by Western blot demonstrating the chimeric proteins native stateand tendency to form a multimer. Untreated samples (i.e., withoutreducing agent or eglycosylation agent) of the FLT3L-Fc-X40L chimericprotein, e.g., control, were loaded into lane 2 in all the blots.Samples in lane 3 were treated with the reducing agent,β-mercaptoethanol. Samples in lane 4 were treated with a deglycosylationagent and the reducing agent. Each individual domain of the chimericprotein was probed using an anti-FLT3L, anti-Fc, or anti-OX40L antibody,respectively. FIG. 5B to FIG. 5D show ELISA data demonstrating thebinding affinity of mFLT3L domain of mFLT3L-Fc-OX40L (FIG. 5B), the ofmFc domain (FIG. 5C), and of the mOX40L domain (FIG. 5D) for theirrespective binding partners.

FIG. 6A shows characterization of a murine FLT3L-Fc-GITRL chimericprotein by Western blot demonstrating the chimeric proteins native stateand tendency to form a multimer. Untreated samples (i.e., withoutreducing agent or deglycosylation agent) of the FLT3L-Fc-GITRL chimericprotein, e.g., control, were loaded into lane 2 in all the blots.Samples in lane 3 were treated with the reducing agent,3-mercaptoethanol. Samples in lane 4 were treated with a deglycosylationagent and the reducing agent. Each individual domain of the chimericprotein was probed using an anti-FLT3L, anti-Fc, or anti-GITRL antibody,respectively. FIG. 6B to FIG. 6D show ELISA data demonstrating thebinding affinity of mFLT3L domain of mFLT3L-Fc-GITRL (FIG. 6B), the ofmFc domain (FIG. 6C), and of the mGITRL domain (FIG. 6D) for theirrespective binding partners.

FIG. 7A compiles the mFLT3L domain ELISA assay data from FIG. 3B, FIG.4B, FIG. 5B, and FIG. 6B. FIG. 7B compiles the mFc domain ELISA datafrom FIG. 3C, FIG. 4C, FIG. 5C, and FIG. 6C.

FIG. 8 shows dual ELISA data of mFLT3L-Fc-OX40L, mFLT3L-Fc-4-1BBL, andFLT3L-Fc-CD40L.

FIG. 9A to FIG. 9E shows characterization of the mFLT3L-Fc-CD40Lchimeric protein. FIG. 9A shows results from the Octet system formeasuring affinity with mCD40-his capture (top curve is mFLT3L-Fc-CD40L,middle curve is mCD40L-Fc, and bottom curve is blank). FIG. 9B showsresults from the Octet system for measuring affinity with mFLT3-hiscapture (top curve is mFLT3L-Fc-CD40L, middle curve is mCD40L-Fc, andbottom curve is blank). Myt sister lives out C shows a summary of thedata of FIG. 9A and FIG. 9B. FIG. 9D shows results of an NFkB-mCD40luciferase reporter assay.

FIG. 9E shows a PathHunter U2OS cell-based assay for CD40L signaling(NFkB activity, non-canonical, top curve is mFLT3L-Fc-CD40L, middlecurve is mCD40L-Fc, and bottom curve is negative control). FIG. 9F showsproliferation of a model cells system (mFLT3 over-expressing Ba/F3cells) in response to Flt3 signaling. The dotted line indicates themaximum proliferation of the untreated cells. Significance wasdetermined using one-way unpaired T-test.

FIG. 10A shows characterization of FLT3L-Fc-GITRL activity with anNFkB-mGITR reporter cell line generated by stably transfecting CHO-K1cells with both a mouse GITR expressing vector and NFkB-luciferasereporter vector (the left bars are mGITRL-Fc, the center bars aremFLT3-Fc-GITRL, and the right bars are mFLT3-Fc-OX40L). FIG. 10B showsproliferation of a model cells system (mFLT3 over-expressing Ba/F3cells) in response to Flt3 signaling via FLT3L-Fc-GITRL. The dotted lineindicates the maximum proliferation of the untreated cells andsignificance was determined using one-way unpaired T-test. FIG. 10Cshows proliferation of a model cells system (mFLT3 over-expressingxxaBa/F3 cells) in response to Flt3 signaling via FLT3L-Fc-OX40L. Thedotted line indicates the maximum proliferation of the untreated cellsand significance was determined using one-way unpaired T-test. FIG. 10Dshows proliferation of a model cells system (mFLT3 over-expressing Ba/F3cells) in response to Flt3 signaling via FLT3L-Fc-4-1BBL. The dottedline indicates the maximum proliferation of the untreated cells andsignificance was determined using one-way unpaired T-test.

FIG. 11 shows in vivo dendritic cell activation by various FLT3L-basedchimeric proteins.

FIG. 12A shows in vivo serum cytokines by various FLT3L-based chimericproteins. Mice were injected for 9 or 11 consecutive days, and thenmesenteric lymph nodes (MLN)/Spleens were isolated on day 10 or 12 andanalyzed by flow cytometry. FIG. 12B shows in vivo serum cytokines byvarious FLT3L-based chimeric proteins. Mice were injected for 9 or 11consecutive days, and then MLN/Spleens were isolated on day 10 or 12 andanalyzed by flow cytometry.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that chimericproteins can be engineered from the extracellular, or effector, regionof FMS like tyrosine kinase 3 ligand (FLT3L) and the extracellular, oreffector, region of a Type II transmembrane protein. These, FLT3L-basedchimeric proteins provide immune activating or co-stimulatory signals,at least in the treatment of cancer.

In embodiments, the present chimeric proteins increase a number ofantigen presenting cells, e.g., dendritic cells. In embodiments, thepresent chimeric proteins enhance antigen presentation, e.g., tumorantigen presentation.

In embodiments, the present chimeric proteins provide a dualco-stimulatory effect on immune cells, e.g., dendritic cells.

In embodiments, the present chimeric proteins enhance cytokineexpression and/or secretion.

In embodiments, the present chimeric proteins provide a contemporaneouseffect of activation of antigen presenting cells, e.g., dendritic cells,and expansion of antigen presenting cells, e.g., dendritic cells. Forinstance, in embodiments, an FLT3-based signal, from the presentchimeric proteins, may increase a number of dendritic cells, and thispopulation may be activated via a stimulatory signal (e.g., CD40L,OX40L, GITRL, LIGHT, CD30L, TRAIL, FasL, APRIL, BAFF, TWEAK, and 4-1BBL,from the present chimeric proteins).

Interestingly, the present inventors have demonstrated this dual actionof the present chimeric proteins without loss of activity of either sideof the present chimeric proteins or, indeed, an increase in signalingactivity by the individual sides of the present chimeric proteins.Stated another way, the present chimeric proteins providecontemporaneous modulation of dendritic cells with a single construct ina manner that does not sacrifice activity and even increases it.

The present chimeric proteins provide advantages including, withoutlimitation, ease of use and ease of production. This is because twodistinct immunotherapy agents are combined into a single product whichmay allow for a single manufacturing process instead of two independentmanufacturing processes. In addition, administration of a single agentinstead of two separate agents allows for easier administration andgreater patient compliance.

Additionally, since a chimeric protein may have two immune-modulatingdomains, it can activate or co-stimulate two distinct immune stimulatorypathways; thus, this dual-action is more likely to provide anyanti-tumor effect in a patient and/or to provide an enhanced anti-tumoreffect in a patient. Moreover, since the methods operate by multipledistinct pathways, they can be efficacious, at least, in patients who donot respond, respond poorly, or become resistant to treatments thattarget one of the pathways. Thus, a patient who is a poor responder totreatments acting via one of the two pathways, can receive a therapeuticbenefit by targeting multiple pathways.

Chimeric Proteins

The chimeric proteins of the present invention comprise an extracellulardomain of FLT3L and an extracellular domain of a Type II transmembraneprotein, each of which has immune stimulatory properties uponanti-cancer immune cells. Thus, the chimeric proteins are designed toenhance, increase, and/or stimulate the transmission of an immunestimulatory signal to the anti-cancer immune cell.

FLT3L is a Type I transmembrane protein that functions as a cytokine andas a growth factor which activates and induces proliferation of immunesystem cells. FLT3L is biologically active in both in its transmembraneform and in its soluble form, which is generated following proteolyticcleavage of the protein's extracellular domain from its transmembranedomain. It has been shown that FLT3L's extracellular domain (residues1-134) comprises its receptor binding site and is sufficient forbioactivity. See, e.g., Savvides et al., “Flt3 ligand structure andunexpected commonalities of helical bundles and cystine knots” NatStruct Biol. 7(6):486-91 (2000).

Without wishing to be bound by theory, unlike most Type I proteins,FLT3L is immune stimulatory and, when paired with a Type II protein,also typically immune stimulatory, provides a dual co-stimulation immuneeffect.

Aspects of the present invention provide a chimeric protein comprising ageneral structure of: N terminus-(a)-(b)-(c)-C terminus, where (a) is afirst domain comprising an extracellular domain of FMS like tyrosinekinase 3 ligand (FLT3L), (b) is a linker adjoining the first domain andthe second domain, e.g., the linker comprising at least one cysteineresidue capable of forming a disulfide bond and/or comprising ahinge-CH2-CH3 Fc domain, and (c) is a second domain comprising anextracellular domain of a Type II transmembrane protein; wherein thelinker connects the first domain and the second domain.

In a chimeric protein of the present invention, the first domain maycomprise substantially the entire extracellular domain of FLT3L and/orthe second domain may comprise substantially the entire extracellulardomain of the Type II transmembrane protein.

In a chimeric protein of the present invention, the first domain and/orthe second domain may be capable of activating an immune stimulatorysignal.

In a chimeric protein of the present invention, the chimeric protein isa recombinant fusion protein, e.g., a single polypeptide having theextracellular domains disclosed herein. For example, in embodiments, thechimeric protein is translated as a single unit in a prokaryotic cell, aeukaryotic cell, or a cell-free expression system.

In embodiments, the present chimeric protein is producible in amammalian host cell as a secretable and fully functional singlepolypeptide chain.

In embodiments, chimeric protein refers to a recombinant protein ofmultiple polypeptides, e.g., multiple extracellular domains disclosedherein, that are combined (via covalent or no-covalent bonding) to yielda single unit, e.g., in vitro (e.g., with one or more synthetic linkersdisclosed herein).

In embodiments, the chimeric protein is chemically synthesized as onepolypeptide or each domain may be chemically synthesized separately andthen combined. In embodiments, a portion of the chimeric protein istranslated and a portion is chemically synthesized.

In embodiments, an extracellular domain refers to a portion of atransmembrane protein which is capable of interacting with theextracellular environment. In embodiments, an extracellular domainrefers to a portion of a transmembrane protein which is sufficient forbinding to a ligand or receptor and is effective in transmitting asignal to a cell. In embodiments, an extracellular domain is the entireamino acid sequence of a transmembrane protein which is normally presentat the exterior of a cell or of the cell membrane. In embodiments, anextracellular domain is that portion of an amino acid sequence of atransmembrane protein which is external of a cell or of the cellmembrane and is needed for signal transduction and/or ligand binding asmay be assayed using methods know in the art (e.g., in vitro ligandbinding and/or cellular activation assays).

Transmembrane proteins typically consist of an extracellular domain, oneor a series of transmembrane domains, and an intracellular domain.Without wishing to be bound by theory, the extracellular domain of atransmembrane protein is responsible for interacting with a solublereceptor or ligand or membrane-bound receptor or ligand (i.e., amembrane of an adjacent cell). Without wishing to be bound by theory,the trans-membrane domain(s) is responsible for localizing thetransmembrane protein to the plasma membrane. Without wishing to bebound by theory, the intracellular domain of a transmembrane protein isresponsible for coordinating interactions with cellular signalingmolecules to coordinate intracellular responses with the extracellularenvironment (or visa-versa).

There are generally two types of single-pass transmembrane proteins:Type I transmembrane proteins which have an extracellular amino terminusand an intracellular carboxy terminus (see, FIG. 1A, left protein) andType II transmembrane proteins which have an extracellular carboxyterminus and an intracellular amino terminus (see, FIG. 1A, rightprotein). Type I and Type II transmembrane proteins can be eitherreceptors or ligands. For Type I transmembrane proteins, the aminoterminus of the protein faces outside the cell, and therefore containsthe functional domains that are responsible for interacting with otherbinding partners (either ligands or receptors) in the extracellularenvironment (see, FIG. 1B, left protein). For Type II transmembraneproteins, the carboxy terminus of the protein faces outside the cell,and therefore contains the functional domains that are responsible forinteracting with other binding partners (either ligands or receptors) inthe extracellular environment (see, FIG. 1B, right protein). Thus, thesetwo types of transmembrane proteins have opposite orientations to eachother relative to the cell membrane.

Chimeric proteins of the present invention comprise an extracellulardomain of FLT3L and an extracellular domain of a Type II transmembraneprotein. Thus, a chimeric protein of the present invention comprises, atleast, a first domain comprising the extracellular domain of FLT3L,which is connected—directly or via a linker—to a second domaincomprising the extracellular domain of a Type II transmembrane protein.As illustrated in FIG. 1C and FIG. 1D, when the domains are linked in anamino-terminal to carboxy-terminal orientation, the first domain islocated on the “left” side of the chimeric protein and is “outwardfacing” and the second domain is located on “right” side of the chimericprotein and is “outward facing”.

Other configurations of first and second domains are envisioned, e.g.,the first domain is outward facing and the second domain is inwardfacing, the first domain is inward facing and the second domain isoutward facing, and the first and second domains are both inward facing.When both domains are “inward facing”, the chimeric protein would havean amino-terminal to carboxy-terminal configuration comprising anextracellular domain of a Type II transmembrane protein, a linker, andan extracellular domain of FLT3L. In such configurations, it may benecessary for the chimeric protein to include extra “slack”, asdescribed elsewhere herein, to permit binding of the chimeric protein toone or both of its receptors/ligands.

Constructs could be produced by cloning the nucleic acids encoding thethree fragments (the extracellular domain of FLT3L, followed by a linkersequence, followed by the extracellular domain of a Type IItransmembrane protein) into a vector (plasmid, viral or other) whereinthe amino terminus of the complete sequence corresponded to the ‘left’side of the molecule containing the extracellular domain of FLT3L andthe carboxy terminus of the complete sequence corresponded to the‘right’ side of the molecule containing the Type II transmembraneprotein. In embodiments of chimeric proteins having one of the otherconfigurations, as described above, a construct would comprise threenucleic acids such that the translated chimeric protein produced wouldhave the desired configuration, e.g., a dual inward-facing chimericprotein. Accordingly, in embodiments, the present chimeric proteins areengineered as such.

Chimeric proteins of the present invention have a first domain which issterically capable of binding its ligand/receptor and/or a second domainwhich is sterically capable of binding its ligand/receptor. This meansthat there is sufficient overall flexibility in the chimeric proteinand/or physical distance between an extracellular domain (or portionthereof) and the rest of the chimeric protein such that theligand/receptor binding domain of the extracellular domain is notsterically hindered from binding its ligand/receptor. This flexibilityand/or physical distance (which is herein referred to as “slack”) may benormally present in the extracellular domain(s), normally present in thelinker, and/or normally present in the chimeric protein (as a whole).Alternately, or additionally, the chimeric protein may be modified byincluding one or more additional amino acid sequences (e.g., the joininglinkers described below) or synthetic linkers (e.g., a polyethyleneglycol (PEG) linker) which provide additional slack needed to avoidsteric hindrance.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of one or more of the immune-modulating agentsdescribed in Mahoney, Nature Reviews Drug Discovery 2015:14; 561-585,the entire contents of which are hereby incorporated by reference.

In embodiments, the chimeric proteins of the present invention comprisevariants of the extracellular domain of FLT3L. As examples, the variantmay have at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with the known amino acidsequence of FLT3L, e.g., human FLT3L.

One of ordinary skill may select variants of the known amino acidsequence of FLT3L by consulting the literature, e.g., Zorn, et al.(2015) “Crystal Structure of the FLT3 Kinase Domain Bound to theInhibitor Quizartinib (AC220).” PLoS ONE 10(4): e0121177, and Graddis,et al. “Structure-Function Analysis of FLT3 Ligand-FLT3 ReceptorInteractions Using a Rapid Functional Screen” The Journal of BiologicalChemistry 273, 17626-17633, each of which is incorporated by referencein its entirety.

In embodiments, the extracellular domain of human FLT3L comprises thefollowing amino acid sequence:

(SEQ ID NO: 57): TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEA TAPTAPQP

In embodiments, the chimeric proteins of the present invention comprisevariants of the extracellular domain of FLT3L. As examples, the variantmay have at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with the known amino acidsequence of SEQ ID NO: 57.

In embodiments, a variant of the extracellular domain of FLT3L comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 57.

In embodiments, the present chimeric proteins increase a number ofantigen presenting cells, e.g., dendritic cells. In embodiments, thepresent chimeric proteins increase a number of activated dendriticcells, e.g., CD11c+ and/or CD103+ dendritic cells.

In embodiments, the present chimeric proteins enhance antigenpresentation, e.g., tumor antigen presentation, e.g., by dendriticcells, e.g., by activated dendritic cells, e.g., CD11c+ and/or CD103+dendritic cells.

In embodiments, the present chimeric proteins provide a dualco-stimulatory effect on immune cells, e.g., antigen presenting cells,e.g., dendritic cells.

In embodiments, the present chimeric proteins enhance cytokineexpression and/or secretion.

In embodiments, the present chimeric proteins provide a contemporaneouseffect of activation of antigen presenting cells, e.g., dendritic cells,and expansion of antigen presenting cells, e.g., dendritic cells. Forinstance, in embodiments, an FLT3-based signal, from the presentchimeric proteins, may increase a number of dendritic cells, and thispopulation may be activated via a stimulatory signal (e.g., CD40L,OX40L, GITRL, LIGHT, CD30L, TRAIL, FasL, APRIL, BAFF, TWEAK, and 4-1BBL,among others from the present chimeric proteins).

In a chimeric protein of the present invention, the Type IItransmembrane protein may be selected from the group consisting of:CD40L, 4-1BBL, APRIL, BAFF, BTNL2, CD28, CD30L, CD70, C-type lectindomain (CLEC) family members, FasL, GITRL, LIGHT, LTa, LTa1b2, NKG2A,NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, and TRAIL. In embodiments, theType II transmembrane protein is 4-1BBL. In embodiments, the Type IItransmembrane protein is CD40L. In embodiments, the Type IItransmembrane protein is CD70. In embodiments, the Type II transmembraneprotein is GITRL. In embodiments, the Type II transmembrane protein isOX40L. In embodiments, the Type II transmembrane protein is TL1A.

In embodiments, the chimeric proteins of the present invention comprisevariants of the extracellular domain of a Type II transmembrane proteindisclosed herein. As examples, the variant may have at least about 60%,or at least about 61%, or at least about 62%, or at least about 63%, orat least about 64%, or at least about 65%, or at least about 66%, or atleast about 67%, or at least about 68%, or at least about 69%, or atleast about 70%, or at least about 71%, or at least about 72%, or atleast about 73%, or at least about 74%, or at least about 75%, or atleast about 76%, or at least about 77%, or at least about 78%, or atleast about 79%, or at least about 80%, or at least about 81%, or atleast about 82%, or at least about 83%, or at least about 84%, or atleast about 85%, or at least about 86%, or at least about 87%, or atleast about 88%, or at least about 89%, or at least about 90%, or atleast about 91%, or at least about 92%, or at least about 93%, or atleast about 94%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% sequenceidentity with the known amino acid sequence of any of the disclosedextracellular domains of a Type II transmembrane protein as disclosedherein, e.g., a human Type II transmembrane protein.

In embodiments, the chimeric proteins of the present invention comprisevariants of the extracellular domain of one of the Type II transmembraneproteins: 4-1BBL, CD40L, CD70, GITRL, OX40L, or TL1A. As examples, thevariant may have at least about 60%, or at least about 61%, or at leastabout 62%, or at least about 63%, or at least about 64%, or at leastabout 65%, or at least about 66%, or at least about 67%, or at leastabout 68%, or at least about 69%, or at least about 70%, or at leastabout 71%, or at least about 72%, or at least about 73%, or at leastabout 74%, or at least about 75%, or at least about 76%, or at leastabout 77%, or at least about 78%, or at least about 79%, or at leastabout 80%, or at least about 81%, or at least about 82%, or at leastabout 83%, or at least about 84%, or at least about 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 89%, or at least about 90%, or at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% sequence identity with the known aminoacid sequence of any of the extracellular domains of 4-1BBL, CD40L,CD70, GITRL, OX40L, or TL1A, e.g., a human extracellular domain of4-1BBL, CD40L, CD70, GITRL, OX40L, or TL1A.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of4-1BBL. In embodiments, a chimeric protein of the present inventioncomprises a variant of the extracellular domain of FLT3L and a variantof the extracellular domain of 4-1BBL.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of 4-1BBL. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity with4-1BBL, e.g., human 4-1BBL.

One of ordinary skill may select variants of the known amino acidsequence of 4-1BBL by consulting the literature, e.g., Goodwin et al.,“Molecular cloning of a ligand for the inducible T cell gene 4-1BB: amember of an emerging family of cytokines with homology to tumornecrosis factor.” Eur. J. Immunol. 23 (10), 2631-2641 (1993); Aldersonet al., “Molecular and biological characterization of human 4-1BB andits ligand.” Eur. J. Immunol. 24 (9), 2219-2227 (1994); and Arch andThompson “4-1BB and Ox40 are members of a tumor necrosis factor(TNF)-nerve growth factor receptor subfamily that bind TNFreceptor-associated factors and activate nuclear factor kappaB.” Mol.Cell. Biol. 18 (1), 558-565 (1998), and Gilbreth et al. Crystalstructure of the human 4-1BB/4-1BBL complex J Biol Chem. 2018 Jun. 22;293(25):9880-9891, each of which is incorporated by reference in itsentirety, which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human 4-1BBL comprises thefollowing amino acid sequence:

(SEQ ID NO: 58): ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLIGGLSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPS PRSE

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of 4-1BBL. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 58.

In embodiments, a variant of the extracellular domain of 4-1BBLcomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 58.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of CD40L.In embodiments, a chimeric protein of the present invention comprises avariant of the extracellular domain of FLT3L and a variant of theextracellular domain of CD40L.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of CD40L. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withCD40L, e.g., human CD40L.

One of ordinary skill may select variants of the known amino acidsequence of CD40L by consulting the literature, e.g., An, et al.Crystallographic and Mutational Analysis of the CD40-CD154 Complex andIts Implications for Receptor Activation, The Journal of BiologicalChemistry 286, 11226-11235, which is incorporated by reference in itsentirety.

In embodiments, the extracellular domain of human CD40L comprises thefollowing amino acid sequence:

(SEQ ID NO: 59): HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVIFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVIDPSQ VSHGTGFTSFGLLKL

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of CD40L. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 59.

In embodiments, a variant of the extracellular domain of CD40L comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 59.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of CD70.In embodiments, a chimeric protein of the present invention comprises avariant of the extracellular domain of FLT3L and a variant of theextracellular domain of 70.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of CD70. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withCD70, e.g., human CD70.

One of ordinary skill may select variants of the known amino acidsequence of CD70 by consulting the literature, e.g., Goodwin et al.,“Molecular and biological characterization of a ligand for CD27 definesa new family of cytokines with homology to tumor necrosis factor.” Cell73 (3), 447-456 (1993), Bowman et al., “The cloning of CD70 and itsidentification as the ligand for CD27” J. Immunol. 152 (4), 1756-1761(1994), Hintzen et al., “CD70 represents the human ligand for CD27” Int.Immunol. 6 (3), 477-480 (1994), and Hintzen et al., “Characterization ofthe human CD27 ligand, a novel member of the TNF gene family” J.Immunol. 152 (4), 1762-1773 (1994), each of which is incorporated byreference in its entirety.

In embodiments, the extracellular domain of human CD70 comprises thefollowing amino acid sequence:

(SEQ ID NO: 60): QRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGV QWVRP

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of CD70. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 60.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of OX40L.In embodiments, a chimeric protein of the present invention comprises avariant of the extracellular domain of FLT3L and a variant of theextracellular domain of OX40L.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of OX40L. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withOX40L, e.g., human OX40L.

One of ordinary skill may select variants of the known amino acidsequence of OX40L by consulting the literature, e.g., CROFT, et al.,“The Significance of OX40 and OX40L to T cell Biology and ImmuneDisease,” Immunol Rev., 229(1), PP. 173-191, 2009 and BAUM, et al.,“Molecular characterization of murine and human OX40/0X40 ligandsystems: identification of a human OX40 ligand as the HTL V-1-regulatedprotein gp34,” The EMBO Journal, Vol. 13, No. 77, PP. 3992-4001, 1994,each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human OX40L comprises thefollowing amino acid sequence:

(SEQ ID NO: 61) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of OX40L. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 61.

In embodiments, a variant of the extracellular domain of OX40L comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 61.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of GITRL.In embodiments, a chimeric protein of the present invention comprises avariant of the extracellular domain of FLT3L and a variant of theextracellular domain of GITRL.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of GITRL. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withGITRL, e.g., human GITRL.

One of ordinary skill may select variants of the known amino acidsequence of GITRL by consulting the literature, e.g., Chattopadhyay etal. “Evolution of GITRL immune function: Murine GITRL exhibits uniquestructural and biochemical properties within the TNF superfamily.” PNAS,Volume 105, Issue 2, 2008, pp. 635-640 and Zjou, et al. “Structuralbasis for ligand-mediated mouse GITR activation Structural basis forligand-mediated mouse GITR activation.” PNAS Jan. 15, 2008. 105 (2)641-645 each of which is incorporated by reference in its entirety.

In embodiments, the extracellular domain of human GITRL comprises thefollowing amino acid sequence:

(SEQ ID NO: 62): ETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of GITRL. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 62.

In embodiments, a variant of the extracellular domain of GITRL comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 62.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L and the extracellular domain of TL1A.In embodiments, a chimeric protein of the present invention comprises avariant of the extracellular domain of FLT3L and a variant of theextracellular domain of TL1A.

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of T1LA. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withTL1A, e.g., human TL1A.

One of ordinary skill may select variants of the known amino acidsequence of TL1A by consulting the literature, e.g., Tan et al.,“Characterization of a novel TNF-like ligand and recently described TNFligand and TNF receptor superfamily genes and their constitutive andinducible expression in hematopoietic and non-hematopoietic cells” Gene204 (1-2), 35-46 (1997), and Zhai et al., “VEGI, a novel cytokine of thetumor necrosis factor family, is an angiogenesis inhibitor thatsuppresses the growth of colon carcinomas in vivo.” FASEB J. 13 (1),181-189 (1999), each of which is incorporated by reference in itsentirety.

In embodiments, the extracellular domain of human TL1A comprises thefollowing amino acid sequence:

(SEQ ID NO: 63): RAQGEACVQFQALKGQEFAPSHQQVYAPLRADGDKPRAHLTWRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITWITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

In embodiments, a chimeric protein used in methods of the presentinvention comprises a variant of the extracellular domain of TL1A. Asexamples, the variant may have at least about 60%, or at least about61%, or at least about 62%, or at least about 63%, or at least about64%, or at least about 65%, or at least about 66%, or at least about67%, or at least about 68%, or at least about 69%, or at least about70%, or at least about 71%, or at least about 72%, or at least about73%, or at least about 74%, or at least about 75%, or at least about76%, or at least about 77%, or at least about 78%, or at least about79%, or at least about 80%, or at least about 81%, or at least about82%, or at least about 83%, or at least about 84%, or at least about85%, or at least about 86%, or at least about 87%, or at least about88%, or at least about 89%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99% sequence identity withSEQ ID NO: 63.

In any herein-disclosed aspect and embodiment, the chimeric protein maycomprise an amino acid sequence having one or more amino acid mutationsrelative to any of the protein sequences disclosed herein. Inembodiments, the one or more amino acid mutations may be independentlyselected from substitutions, insertions, deletions, and truncations.

In embodiments, the amino acid mutations are amino acid substitutions,and may include conservative and/or non-conservative substitutions.“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As usedherein, “conservative substitutions” are defined as exchanges of anamino acid by another amino acid listed within the same group of the sixstandard amino acid groups shown above. For example, the exchange of Aspby Glu retains one negative charge in the so modified polypeptide. Inaddition, glycine and proline may be substituted for one another basedon their ability to disrupt α-helices. As used herein, “non-conservativesubstitutions” are defined as exchanges of an amino acid by anotheramino acid listed in a different group of the six standard amino acidgroups (1) to (6) shown above.

In embodiments, the substitutions may also include non-classical aminoacids (e.g., selenocysteine, pyrrolysine, N-formylmethionine β-alanine,GABA and 5-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme,citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Mutations may also be made to the nucleotide sequences of the chimericproteins by reference to the genetic code, including taking into accountcodon degeneracy.

In embodiments, a chimeric protein is capable of binding murineligand(s)/receptor(s).

In embodiments, a chimeric protein is capable of binding humanligand(s)/receptor(s).

In embodiments, each extracellular domain (or variant thereof) of thechimeric protein binds to its cognate receptor or ligand with a K_(D) ofabout 1 nM to about 5 nM, for example, about 1 nM, about 1.5 nM, about 2nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, orabout 5 nM. In embodiments, the chimeric protein binds to a cognatereceptor or ligand with a K_(D) of about 5 nM to about 15 nM, forexample, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM,about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about10 nM, about 10.5 nM, about 11 nM, about 11.5 nM, about 12 nM, about12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, orabout 15 nM.

In embodiments, each extracellular domain (or variant thereof) of thechimeric protein binds to its cognate receptor or ligand with a K_(D) ofless than about 1 pM, about 900 nM, about 800 nM, about 700 nM, about600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about150 nM, about 130 nM, about 100 nM, about 90 nM, about 80 nM, about 70nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM,about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about10 nM, or about 5 nM, or about 1 nM (as measured, for example, bysurface plasmon resonance or biolayer interferometry). In embodiments,the chimeric protein binds to human CSF1 with a K_(D) of less than about1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM,about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM,about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, bysurface plasmon resonance or biolayer interferometry).

As used herein, a variant of an extracellular domain is capable ofbinding the receptor/ligand of a native extracellular domain. Forexample, a variant may include one or more mutations in an extracellulardomain which do not affect its binding affinity to its receptor/ligand;alternately, the one or more mutations in an extracellular domain mayimprove binding affinity for the receptor/ligand; or the one or moremutations in an extracellular domain may reduce binding affinity for thereceptor/ligand, yet not eliminate binding altogether. In embodiments,the one or more mutations are located outside the binding pocket wherethe extracellular domain interacts with its receptor/ligand. Inembodiments, the one or more mutations are located inside the bindingpocket where the extracellular domain interacts with itsreceptor/ligand, as long as the mutations do not eliminate bindingaltogether. Based on the skilled artisan's knowledge and the knowledgein the art regarding receptor-ligand binding, s/he would know whichmutations would permit binding and which would eliminate binding.

In embodiments, the chimeric protein exhibits enhanced stability,high-avidity binding characteristics, prolonged off-rate for targetbinding and protein half-life relative to single-domain fusion proteinor antibody controls.

A chimeric protein of the present invention may comprise more than twoextracellular domains. For example, the chimeric protein may comprisethree, four, five, six, seven, eight, nine, ten, or more extracellulardomains. A second extracellular domain may be separated from a thirdextracellular domain via a linker, as disclosed herein. Alternately, asecond extracellular domain may be directly linked (e.g., via a peptidebond) to a third extracellular domain. In embodiments, a chimericprotein includes extracellular domains that are directly linked andextracellular domains that are indirectly linked via a linker, asdisclosed herein.

Linkers

In embodiments, the chimeric protein comprises a linker.

In embodiments, the linker comprising at least one cysteine residuecapable of forming a disulfide bond. The at least one cysteine residueis capable of forming a disulfide bond between a pair (or more) ofchimeric proteins. Without wishing to be bound by theory, such disulfidebond forming is responsible for maintaining a useful multimeric state ofchimeric proteins. This allows for efficient production of the chimericproteins; it allows for desired activity in vitro and in vivo.

In a chimeric protein of the present invention, the linker is apolypeptide selected from a flexible amino acid sequence, an IgG hingeregion, or an antibody sequence.

In embodiments, the linker is derived from naturally-occurringmulti-domain proteins or is an empirical linker as described, forexample, in Chichili et al, (2013), Protein Sci. 22(2):153-167, Chen etal, (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents ofwhich are hereby incorporated by reference. In embodiments, the linkermay be designed using linker designing databases and computer programssuch as those described in Chen et al, (2013), Adv Drug Deliv Rev.65(10):1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312,the entire contents of which are hereby incorporated by reference.

In embodiments, the linker comprises a polypeptide. In embodiments, thepolypeptide is less than about 500 amino acids long, about 450 aminoacids long, about 400 amino acids long, about 350 amino acids long,about 300 amino acids long, about 250 amino acids long, about 200 aminoacids long, about 150 amino acids long, or about 100 amino acids long.For example, the linker may be less than about 100, about 95, about 90,about 85, about 80, about 75, about 70, about 65, about 60, about 55,about 50, about 45, about 40, about 35, about 30, about 25, about 20,about 19, about 18, about 17, about 16, about 15, about 14, about 13,about 12, about 11, about 10, about 9, about 8, about 7, about 6, about5, about 4, about 3, or about 2 amino acids long.

In embodiments, the linker is flexible.

In embodiments, the linker is rigid.

In embodiments, the linker is substantially comprised of glycine andserine residues (e.g., about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).

In embodiments, the linker comprises a hinge region of an antibody(e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1,IgG2, IgG3, and IgG4, and IgA1, and IgA2)). The hinge region, found inIgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer,allowing the Fab portion to move freely in space. In contrast to theconstant regions, the hinge domains are structurally diverse, varying inboth sequence and length among immunoglobulin classes and subclasses.For example, the length and flexibility of the hinge region varies amongthe IgG subclasses. The hinge region of IgG1 encompasses amino acids216-231 and, because it is freely flexible, the Fab fragments can rotateabout their axes of symmetry and move within a sphere centered at thefirst of two inter-heavy chain disulfide bridges. IgG2 has a shorterhinge than IgG1, with 12 amino acid residues and four disulfide bridges.The hinge region of IgG2 lacks a glycine residue, is relatively short,and contains a rigid poly-proline double helix, stabilized by extrainter-heavy chain disulfide bridges. These properties restrict theflexibility of the IgG2 molecule. IgG3 differs from the other subclassesby its unique extended hinge region (about four times as long as theIgG1 hinge), containing 62 amino acids (including 21 prolines and 11cysteines), forming an inflexible poly-proline double helix. In IgG3,the Fab fragments are relatively far away from the Fc fragment, givingthe molecule a greater flexibility. The elongated hinge in IgG3 is alsoresponsible for its higher molecular weight compared to the othersubclasses. The hinge region of IgG4 is shorter than that of IgG1 andits flexibility is intermediate between that of IgG1 and IgG2. Theflexibility of the hinge regions reportedly decreases in the orderIgG3>IgG1>IgG4>IgG2. In embodiments, the linker may be derived fromhuman IgG4 and contain one or more mutations to enhance dimerization(including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of C_(H1) to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In embodiments, the linker of the presentinvention comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)).

In a chimeric protein of the present invention, the linker comprises ahinge-CH2-CH3 Fc domain derived from IgG4. In embodiments, the linkercomprises a hinge-CH2-CH3 Fc domain derived from a human IgG4. Inembodiments, the linker comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO: 3, e.g., at least 95% identical to the amino acidsequence of SEQ ID NO: 2. In embodiments, the linker comprises one ormore joining linkers, such joining linkers independently selected fromSEQ ID NOs: 4-50 (or a variant thereof). In embodiments, the linkercomprises two or more joining linkers each joining linker independentlyselected from SEQ ID NOs: 4-50 (or a variant thereof); wherein onejoining linker is N terminal to the hinge-CH2-CH3 Fc domain and anotherjoining linker is C terminal to the hinge-CH2-CH3 Fc domain.

In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derivedfrom a human IgG1 antibody. In embodiments, the Fc domain exhibitsincreased affinity for and enhanced binding to the neonatal Fc receptor(FcRn).

In embodiments, the Fc domain includes one or more mutations thatincreases the affinity and enhances binding to FcRn. Without wishing tobe bound by theory, it is believed that increased affinity and enhancedbinding to FcRn increases the in vivo half-life of the present chimericproteins.

In embodiments, the Fc domain in a linker contains one or more aminoacid substitutions at amino acid residue 250, 252, 254, 256, 308, 309,311, 416, 428, 433 or 434 (in accordance with Kabat numbering, as in asin Kabat, et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991) expressly incorporated herein by reference), or equivalentsthereof. In embodiments, the amino acid substitution at amino acidresidue 250 is a substitution with glutamine. In embodiments, the aminoacid substitution at amino acid residue 252 is a substitution withtyrosine, phenylalanine, tryptophan or threonine. In embodiments, theamino acid substitution at amino acid residue 254 is a substitution withthreonine. In embodiments, the amino acid substitution at amino acidresidue 256 is a substitution with serine, arginine, glutamine, glutamicacid, aspartic acid, or threonine. In embodiments, the amino acidsubstitution at amino acid residue 308 is a substitution with threonine.In embodiments, the amino acid substitution at amino acid residue 309 isa substitution with proline. In embodiments, the amino acid substitutionat amino acid residue 311 is a substitution with serine. In embodiments,the amino acid substitution at amino acid residue 385 is a substitutionwith arginine, aspartic acid, serine, threonine, histidine, lysine,alanine or glycine. In embodiments, the amino acid substitution at aminoacid residue 386 is a substitution with threonine, proline, asparticacid, serine, lysine, arginine, isoleucine, or methionine. Inembodiments, the amino acid substitution at amino acid residue 387 is asubstitution with arginine, proline, histidine, serine, threonine, oralanine. In embodiments, the amino acid substitution at amino acidresidue 389 is a substitution with proline, serine or asparagine. Inembodiments, the amino acid substitution at amino acid residue 416 is asubstitution with serine. In embodiments, the amino acid substitution atamino acid residue 428 is a substitution with leucine. In embodiments,the amino acid substitution at amino acid residue 433 is a substitutionwith arginine, serine, isoleucine, proline, or glutamine. Inembodiments, the amino acid substitution at amino acid residue 434 is asubstitution with histidine, phenylalanine, or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constantregion) comprises one or more mutations such as substitutions at aminoacid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabatnumbering, as in as in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) expressly incorporated hereinby reference). In embodiments, the IgG constant region includes a tripleM252Y/S254T/T256E mutation or YTE mutation. In embodiments, the IgGconstant region includes a triple H433K/N434F/Y436H mutation or KFHmutation. In embodiments, the IgG constant region includes an YTE andKFH mutation in combination.

In embodiments, the linker comprises an IgG constant region thatcontains one or more mutations at amino acid residues 250, 253, 307,310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, asin as in Kabat, et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991) expressly incorporated herein by reference). Illustrativemutations include T250Q, M428L, T307A, E380A, 1253A, H310A, M428L,H433K, N434A, N434F, N434S, and H435A. In embodiments, the IgG constantregion comprises a M428L/N434S mutation or LS mutation. In embodiments,the IgG constant region comprises a T250Q/M428L mutation or QL mutation.In embodiments, the IgG constant region comprises an N434A mutation. Inembodiments, the IgG constant region comprises a T307A/E380A/N434Amutation or AAA mutation. In embodiments, the IgG constant regioncomprises an 1253A/H310A/H435A mutation or IHH mutation. In embodiments,the IgG constant region comprises a H433K/N434F mutation. Inembodiments, the IgG constant region comprises a M252Y/S254T/T256E and aH433K/N434F mutation in combination.

Additional exemplary mutations in the IgG constant region are described,for example, in Robbie, et al, Antimicrobial Agents and Chemotherapy(2013), 57(12):6147-6153, Dall'Acqua et al, JBC (2006),281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002),169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys et al. Journalof Immunology. (2015), 194(11):5497-508, and U.S. Pat. No. 7,083,784,the entire contents of which are hereby incorporated by reference.

An illustrative Fc stabilizing mutant is S228P. Illustrative Fchalf-life extending mutants are T250Q, M428L, V308T, L309P, and Q311Sand the present linkers may comprise 1, or 2, or 3, or 4, or 5 of thesemutants.

In embodiments, the chimeric protein binds to FcRn with high affinity.In embodiments, the chimeric protein may bind to FcRn with a K_(D) ofabout 1 nM to about 80 nM. For example, the chimeric protein may bind toFcRn with a K_(D) of about 1 nM, about 2 nM, about 3 nM, about 4 nM,about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM,about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM,about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80nM. In embodiments, the chimeric protein may bind to FcRn with a K_(D)of about 9 nM. In embodiments, the chimeric protein does notsubstantially bind to other Fc receptors (i.e. other than FcRn) witheffector function.

In embodiments, the Fc domain in a linker has the amino acid sequence ofSEQ ID NO: 1 (see Table 1, below), or at least 90%, or 93%, or 95%, or97%, or 98%, or 99% identity thereto. In embodiments, mutations are madeto SEQ ID NO: 1 to increase stability and/or half-life. For instance, inembodiments, the Fc domain in a linker comprises the amino acid sequenceof SEQ ID NO: 2 (see Table 1, below), or at least 90%, or 93%, or 95%,or 97%, or 98%, or 99% identity thereto. For instance, in embodiments,the Fc domain in a linker comprises the amino acid sequence of SEQ IDNO: 3 (see Table 1, below), or at least 90%, or 93%, or 95%, or 97%, or98%, or 99% identity thereto.

Further, one or more joining linkers may be employed to connect an Fcdomain in a linker (e.g., one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3 or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto) and the extracellular domains. For example, any one of SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or variants thereof may connect an extracellular domain asdisclosed herein and an Fc domain in a linker as disclosed herein.Optionally, any one of SEQ ID NOs: 4 to 50, or variants thereof arelocated between an extracellular domain as disclosed herein and an Fcdomain as disclosed herein.

In embodiments, the present chimeric proteins may comprise variants ofthe joining linkers disclosed in Table 1, below. For instance, a linkermay have at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 4 to 50.

In embodiments, the first and second joining linkers may be different orthey may be the same.

Without wishing to be bound by theory, including a linker comprising atleast a part of an Fc domain in a chimeric protein, helps avoidformation of insoluble and, likely, non-functional protein concatenatedoligomers and/or aggregates. This is in part due to the presence ofcysteines in the Fc domain which are capable of forming disulfide bondsbetween chimeric proteins.

In embodiments, a chimeric protein may comprise one or more joininglinkers, as disclosed herein, and lack a Fc domain linker, as disclosedherein.

In embodiments, the first and/or second joining linkers areindependently selected from the amino acid sequences of SEQ ID NOs: 4 to50 and are provided in Table 1 below:

TABLE 1 Illustravtive linkers (Fc domain linkers and joining linkers)SEQ ID NO. Sequence  1APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK  2APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTTPHSDWLSGKEYKCKVSSKGLPSSEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK  3APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK  4 SKYGPPCPSCP  5 SKYGPPCPPCP  6 SKYGPP  7 IEGRMD  8GGGVPRDCG  9 IEGRMDGGGGAGGGG 10 GGGSGGGS 11 GGGSGGGGSGGG 12EGKSSGSGSESKST 13 GGSG 14 GGSGGGSGGGSG 15 EAAAKEAAAKEAAAK 16EAAAREAAAREAAAREAAAR 17 GGGGSGGGGSGGGGSAS 18 GGGGAGGGG 19GS or GGS or LE 20 GSGSGS 21 GSGSGSGSGS 22 GGGGSAS 23APAPAPAPAPAPAPAPAPAP 24 CPPC 25 GGGGS 26 GGGGSGGGGS 27 GGGGSGGGGSGGGGS28 GGGGSGGGGSGGGGSGGGGS 29 GGGGSGGGGSGGGGSGGGGSGGGGS 30GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 31 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 32GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 33 GGSGGSGGGGSGGGGS 34 GGGGGGGG35 GGGGGG 36 EAAAK 37 EAAAKEAAAK 38 EAAAKEAAAKEAAAK 39 AEAAAKEAAAKA 40AEAAAKEAAAKEAAAKA 41 AEAAAKEAAAKEAAAKEAAAKA 42AEAAAKEAAAKEAAAKEAAAKEAAAKA 43AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA 44 PAPAP 45KESGSVSSEQLAQFRSLD 46 GSAGSAAGSGEF 47 GGGSE 48 GSESG 49 GSEGS 50GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS

In embodiments, the joining linker substantially comprises glycine andserine residues (e.g., about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).For example, in embodiments, the joining linker is (Gly₄Ser)_(n), wheren is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ IDNO: 25 to SEQ ID NO: 32, respectively). In embodiments, the joininglinker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33). Additionalillustrative joining linkers include, but are not limited to, linkershaving the sequence LE, (EAAAK)_(n) (n=1-3) (SEQ ID NO: 36 to SEQ ID NO:38), A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 39 to SEQ ID NO: 42),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44),KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu. Inembodiments, the joining linker is GGS. In embodiments, a joining linkerhas the sequence (Gly), where n is any number from 1 to 100, forexample: (Gly)₈ (SEQ ID NO: 34) and (Gly)₆ (SEQ ID NO: 35).

In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO:47), GSESG (SEQ ID NO: 48), GSEGS (SEQ ID NO: 49),GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and a joininglinker of randomly placed G, S, and E every 4 amino acid intervals.

In embodiments, where a chimeric protein comprises an extracellulardomain (ECD) of FLT3L, one joining linker preceding an Fc domain, asecond joining linker following the Fc domain, and an ECD of a Type IItransmembrane protein, the chimeric protein may comprise the followingstructure:

-   -   ECD of FLT3L-Joining Linker 1-Fc Domain-Joining Linker 2-ECD of        Type II protein

The combination of a first joining linker, an Fc Domain linker, and asecond joining linker is referend to herein as a “modular linker”. Inembodiments, a chimeric protein comprises a modular linker as shown inTable 2:

TABLE 2 Illustrative modular linkers Joining Modular Linker =Joining Linker Joining Linker 1 Fc Linker 2 1 + Fc + Joining Linker 2SKYGPPCPSCP APEFLGGPSVFLFPPKPKDTLMIS IEGRMD SKYGPPCPSCPAPEFLGGPSVFL(SEQ ID NO: 4) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDTLMISRTPEVTCVVVDV WYVDGVEVHNAKTKPREEQFNS SQEDPEVQFNWYVDGVEVHNAKTYRWSVLTVLHQDWLSGKEYKC TKPREEQFNSTYRVVSVLTVLHQ KVSSKGLPSSIEKTISNATGQPREDWLSGKEYKCKVSSKGLPSSIEK PQVYTLPPSQEEMTKNQVSLTCL TISNATGQPREPQVYTLPPSQEEVKGFYPSDIAVEWESNGQPENNY MTKNQVSLTCLVKGFYPSDIAVE KTTPPVLDSDGSFFLYSRLTVDKSWESNGQPENNYKTTPPVLDSDG SWQEGNVFSCSVMHEALHNHYT SFFLYSRLTVDKSSWQEGNVFSCQKSLSLSLGK (SEQ ID NO: 1) SVMHEALHNHYTQKSLSLSLGKIE GRMD (SEQ ID NO: 51)SKYGPPCPSCP APEFLGGPSVFLFPPKPKDQLMIS IEGRMD SKYGPPCPSCPAPEFLGGPSVFL(SEQ ID NO: 4) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDQLMISRTPEVTCVVVD WYVDGVEVHNAKTKPREEQFNS VSQEDPEVQFNWYVDGVEVHNATYRWSVLTTPHSDWLSGKEYKC KTKPREEQFNSTYRVVSVLTTPH KVSSKGLPSSIEKTISNATGQPRESDWLSGKEYKCKVSSKGLPSSIE PQVYTLPPSQEEMTKNQVSLTCL KTISNATGQPREPQVYTLPPSQEVKGFYPSDIAVEWESNGQPENNY EMTKNQVSLTCLVKGFYPSDIAV KTTPPVLDSDGSFFLYSRLTVDKSEWESNGQPENNYKTTPPVLDSD SWQEGNVFSCSVLHEALHNHYT GSFFLYSRLTVDKSSWQEGNVFSQKSLSLSLGK (SEQ ID NO: 2) CSVLHEALHNHYTQKSLSLSLGKI EGRMD (SEQ ID NO: 52)SKYGPPCPSCP APEFLGGPSVFLFPPKPKDQLMIS IEGRMD SKYGPPCPSCPAPEFLGGPSVFL(SEQ ID NO: 4) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDQLMISRTPEVTCVVVD WYVDGVEVHNAKTKPREEQFNS VSQEDPEVQFNWYVDGVEVHNATYRWSVLTVLHQDWLSGKEYKC KTKPREEQFNSTYRVVSVLTVLH KVSSKGLPSSIEKTISNATGQPREQDWLSGKEYKCKVSSKGLPSSIE PQVYTLPPSQEEMTKNQVSLTCL KTISNATGQPREPQVYTLPPSQEVKGFYPSDIAVEWESNGQPENNY EMTKNQVSLTCLVKGFYPSDIAV KTTPPVLDSDGSFFLYSRLTVDKSEWESNGQPENNYKTTPPVLDSD RWQEGNVFSCSVLHEALHNHYT GSFFLYSRLTVDKSRWQEGNVFSQKSLSLSLGK (SEQ ID NO: 3) CSVLHEALHNHYTQKSLSLSLGKI EGRMD (SEQ ID NO: 53)SKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMIS IEGRMD SKYGPPCPPCPAPEFLGGPSVFL(SEQ ID NO: 5) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDTLMISRTPEVTCVVVDV WYVDGVEVHNAKTKPREEQFNS SQEDPEVQFNWYVDGVEVHNAKTYRWSVLTVLHQDWLSGKEYKC TKPREEQFNSTYRVVSVLTVLHQ KVSSKGLPSSIEKTISNATGQPREDWLSGKEYKCKVSSKGLPSSIEK PQVYTLPPSQEEMTKNQVSLTCL TISNATGQPREPQVYTLPPSQEEVKGFYPSDIAVEWESNGQPENNY MTKNQVSLTCLVKGFYPSDIAVE KTTPPVLDSDGSFFLYSRLTVDKSWESNGQPENNYKTTPPVLDSDG SWQEGNVFSCSVMHEALHNHYT SFFLYSRLTVDKSSWQEGNVFSCQKSLSLSLGK (SEQ ID NO: 1) SVMHEALHNHYTQKSLSLSLGKIE GRMD (SEQ ID NO: 54)SKYGPPCPPCP APEFLGGPSVFLFPPKPKDQLMIS IEGRMD SKYGPPCPPCPAPEFLGGPSVFL(SEQ ID NO: 5) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDQLMISRTPEVTCVVVD WYVDGVEVHNAKTKPREEQFNS VSQEDPEVQFNWYVDGVEVHNATYRWSVLTTPHSDWLSGKEYKC KTKPREEQFNSTYRVVSVLTTPH KVSSKGLPSSIEKTISNATGQPRESDWLSGKEYKCKVSSKGLPSSIE PQVYTLPPSQEEMTKNQVSLTCL KTISNATGQPREPQVYTLPPSQEVKGFYPSDIAVEWESNGQPENNY EMTKNQVSLTCLVKGFYPSDIAV KTTPPVLDSDGSFFLYSRLTVDKSEWESNGQPENNYKTTPPVLDSD SWQEGNVFSCSVLHEALHNHYT GSFFLYSRLTVDKSSWQEGNVFSQKSLSLSLGK (SEQ ID NO: 2) CSVLHEALHNHYTQKSLSLSLGKI EGRMD (SEQ ID NO: 55)SKYGPPCPPCP APEFLGGPSVFLFPPKPKDQLMIS IEGRMD SKYGPPCPPCPAPEFLGGPSVFL(SEQ ID NO: 5) RTPEVTCVVVDVSQEDPEVQFN (SEQ ID NO: 7)FPPKPKDQLMISRTPEVTCVVVD WYVDGVEVHNAKTKPREEQFNS VSQEDPEVQFNWYVDGVEVHNATYRWSVLTVLHQDWLSGKEYKC KTKPREEQFNSTYRVVSVLTVLH KVSSKGLPSSIEKTISNATGQPREQDWLSGKEYKCKVSSKGLPSSIE PQVYTLPPSQEEMTKNQVSLTCL KTISNATGQPREPQVYTLPPSQEVKGFYPSDIAVEWESNGQPENNY EMTKNQVSLTCLVKGFYPSDIAV KTTPPVLDSDGSFFLYSRLTVDKSEWESNGQPENNYKTTPPVLDSD RWQEGNVFSCSVLHEALHNHYT GSFFLYSRLTVDKSRWQEGNVFSQKSLSLSLGK (SEQ ID NO: 3) CSVLHEALHNHYTQKSLSLSLGKI EGRMD (SEQ ID NO: 56)

In embodiments, the present chimeric proteins may comprise variants ofthe modular linkers disclosed in Table 2, above. For instance, a linkermay have at least about 60%, or at least about 61%, or at least about62%, or at least about 63%, or at least about 64%, or at least about65%, or at least about 66%, or at least about 67%, or at least about68%, or at least about 69%, or at least about 70%, or at least about71%, or at least about 72%, or at least about 73%, or at least about74%, or at least about 75%, or at least about 76%, or at least about77%, or at least about 78%, or at least about 79%, or at least about80%, or at least about 81%, or at least about 82%, or at least about83%, or at least about 84%, or at least about 85%, or at least about86%, or at least about 87%, or at least about 88%, or at least about89%, or at least about 90%, or at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% sequence identity with the amino acidsequence of any one of SEQ ID NOs: 51 to 56.

In embodiments, the linker may be flexible, including without limitationhighly flexible. In embodiments, the linker may be rigid, includingwithout limitation a rigid alpha helix. Characteristics of illustrativejoining linkers is shown below in Table 3:

TABLE 3 Characteristics of illustrative joining linkersJoining Linker Sequence Characteristics SKYGPPCPPCP (SEQ ID NO: 5)IgG4 Hinge Region IEGRMD (SEQ ID NO: 7) Linker GGGVPRDCG (SEQ ID NO: 8)Flexible GGGSGGGS (SEQ ID NO: 10) Flexible GGGSGGGGSGGG (SEQ ID NO: 11)Flexible EGKSSGSGSESKST (SEQ ID NO: 12) Flexible + solubleGGSG (SEQ ID NO: 13) Flexible GGSGGGSGGGSG (SEQ ID NO: 14) FlexibleEAAAKEAAAKEAAAK (SEQ ID NO: 15) Rigid Alpha HelixEAAAREAAAREAAAREAAAR (SEQ ID NO: 16) Rigid Alpha HelixGGGGSGGGGSGGGGSAS (SEQ ID NO: 17) Flexible GGGGAGGGG (SEQ ID NO: 18)Flexible GS (SEQ ID NO: 19) Highly flexible GSGSGS (SEQ ID NO: 20)Highly flexible GSGSGSGSGS (SEQ ID NO: 21) Highly flexibleGGGGSAS (SEQ ID NO: 22) Flexible APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 23)Rigid

In embodiments, the linker may be functional. For example, withoutlimitation, the linker may function to improve the folding and/orstability, improve the expression, improve the pharmacokinetics, and/orimprove the bioactivity of the present chimeric protein. In anotherexample, the linker may function to target the chimeric protein to aparticular cell type or location.

In embodiments, a chimeric protein comprises only one joining linkers.

In embodiments, a chimeric protein lacks joining linkers.

In embodiments, the linker is a synthetic linker such as polyethyleneglycol (PEG).

In embodiments, a chimeric protein has a first domain which issterically capable of binding its ligand/receptor and/or the seconddomain which is sterically capable of binding its ligand/receptor. Thus,there is enough overall flexibility in the chimeric protein and/orphysical distance between an extracellular domain (or portion thereof)and the rest of the chimeric protein such that the ligand/receptorbinding domain of the extracellular domain is not sterically hinderedfrom binding its ligand/receptor. This flexibility and/or physicaldistance (which is referred to as “slack”) may be normally present inthe extracellular domain(s), normally present in the linker, and/ornormally present in the chimeric protein (as a whole). Alternately, oradditionally, an amino acid sequence (for example) may be added to oneor more extracellular domains and/or to the linker to provide the slackneeded to avoid steric hindrance. Any amino acid sequence that providesslack may be added. In embodiments, the added amino acid sequencecomprises the sequence (Gly), where n is any number from 1 to 100.Additional examples of addable amino acid sequence include the joininglinkers described in Table 1 and Table 3. In embodiments, a polyethyleneglycol (PEG) linker may be added between an extracellular domain and alinker to provide the slack needed to avoid steric hindrance. Such PEGlinkers are well known in the art.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of 4-1BBL (or a variant thereof). Inembodiments, the linker comprises a hinge-CH2-CH3 Fc domain (or avariant thereof), e.g., from an IgG1 or from IgG4, including human IgG1or IgG4. Thus, in embodiments, a chimeric protein of the presentinvention comprises the extracellular domain of FLT3L (or a variantthereof), linker comprising a hinge-CH2-CH3 Fc domain (or a variantthereof), and the extracellular domain of 4-1BBL (or a variant thereof).Such a chimeric protein may be referred to herein as “FLT3L-Fc-4-1BBL”.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of CD40L (or a variant thereof). Inembodiments, the linker comprises a hinge-CH2-CH3 Fc domain (or avariant thereof), e.g., from an IgG1 or from IgG4, including human IgG1or IgG4. Thus, in embodiments, a chimeric protein of the presentinvention comprises the extracellular domain of FLT3L (or a variantthereof), linker comprising a hinge-CH2-CH3 Fc domain (or a variantthereof), and the extracellular domain of CD40L (or a variant thereof).Such a chimeric protein may be referred to herein as “FLT3L-Fc-CD40L”.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of CD70 (or a variant thereof). In embodiments,the linker comprises a hinge-CH2-CH3 Fc domain (or a variant thereof),e.g., from an IgG1 or from IgG4, including human IgG1 or IgG4.

Thus, in embodiments, a chimeric protein of the present inventioncomprises the extracellular domain of FLT3L (or a variant thereof),linker comprising a hinge-CH2-CH3 Fc domain (or a variant thereof), andthe extracellular domain of CD40L (or a variant thereof). Such achimeric protein may be referred to herein as “FLT3L-Fc-CD70L”.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of GITRL (or a variant thereof). Inembodiments, the linker comprises a hinge-CH2-CH3 Fc domain (or avariant thereof), e.g., from an IgG1 or from IgG4, including human IgG1or IgG4. Thus, in embodiments, a chimeric protein of the presentinvention comprises the extracellular domain of FLT3L (or a variantthereof), linker comprising a hinge-CH2-CH3 Fc domain (or a variantthereof), and the extracellular domain of GITRL (or a variant thereof).Such a chimeric protein may be referred to herein as “FLT3L-Fc-GITRL”.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of OX40L (or a variant thereof). Inembodiments, the linker comprises a hinge-CH2-CH3 Fc domain (or avariant thereof), e.g., from an IgG1 or from IgG4, including human IgG1or IgG4. Thus, in embodiments, a chimeric protein of the presentinvention comprises the extracellular domain of FLT3L (or a variantthereof), linker comprising a hinge-CH2-CH3 Fc domain (or a variantthereof), and the extracellular domain of OX40L (or a variant thereof).Such a chimeric protein may be referred to herein as “FLT3L-Fc-OX40L”.

In embodiments, a chimeric protein of the present invention comprisesthe extracellular domain of FLT3L (or a variant thereof), a linker, andthe extracellular domain of TL1A (or a variant thereof). In embodiments,the linker comprises a hinge-CH2-CH3 Fc domain (or a variant thereof),e.g., from an IgG1 or from IgG4, including human IgG1 or IgG4. Thus, inembodiments, a chimeric protein of the present invention comprises theextracellular domain of FLT3L (or a variant thereof), linker comprisinga hinge-CH2-CH3 Fc domain (or a variant thereof), and the extracellulardomain of CD40L (or a variant thereof). Such a chimeric protein may bereferred to herein as “FLT3L-Fc-TL1A”.

Diseases; Methods of Treatment, and Mechanisms of Action

A chimeric protein disclosed herein may be used in the treatment ofcancer and/or in the treatment of an inflammatory disease, e.g., due toviral infection.

Aspects of the present invention provide methods of treating cancer. Themethods comprise a step of administering to a subject in need thereof aneffective amount of a pharmaceutical composition which comprises achimeric protein as disclosed herein.

It is often desirable to enhance immune stimulatory signal transmissionto boost an immune response, for instance to enhance a patient'santi-tumor immune response.

In embodiments, the chimeric protein of the present invention and/orchimeric protein used in methods of the present invention comprises anextracellular domain of FLT3L and an extracellular domain of a Type IImembrane protein, each of which have immune stimulatory properties.Thus, the binding of the extracellular domain of FLT3L with itsligand/receptor (e.g., FLT3) will enhance, increase, and/or stimulatethe transmission of an immune stimulatory signal.

The chimeric protein of the present invention and/or chimeric proteinused in methods of the present invention further comprises anextracellular domain of a Type II membrane protein which provides animmune stimulatory signal; the Type II membrane protein being, withoutlimitation, is one or more of 4-1BBL, APRIL, BAFF, BTNL2, CD28, CD30L,CD40L, CD70, C-type lectin domain (CLEC) family members, FasL, GITRL,LIGHT, LTa, LTa1b2, NKG2A, NKG2C, NKG2D, OX40L, RANKL, TL1A, TNFa, andTRAIL. Thus, the chimeric protein is engineered to enhances, increases,and/or stimulates the transmission of an immune stimulatory signal, byway of non-limiting example, the binding of one of 4-1BBL, APRIL, BAFF,BTNL2, CD28, CD30L, CD40L, CD70, C-type lectin domain (CLEC) familymembers, FasL, GITRL, LIGHT, LTa, LTa1b2, NKG2A, NKG2C, NKG2D, OX40L,RANKL, TL1A, TNFa, and TRAIL with its ligand/receptor. Accordingly, inembodiments, a chimeric protein has “dual costimulatory” capabilitiesfrom each of its first domain and its second domain.

In embodiments, the chimeric protein comprises an immune stimulatorysignal which is an extracellular domain of a ligand of an immunestimulatory signal and this acts on a T cell that bears a cognatereceptor of the immune stimulatory signal.

In embodiments, the extracellular domain may be used to provideartificial signaling.

In embodiments, the extracellular domain of a Type II transmembraneprotein is an immune stimulatory signal.

In embodiments, the present invention pertains to cancers and/or tumors;for example, the treatment or prevention of cancers and/or tumors. Asdisclosed elsewhere herein, the treatment of cancer involves, inembodiments, modulating the immune system with the present chimericproteins to favor of increasing or activating immune stimulatorysignals. In embodiments, the method reduces the amount or activity ofregulatory T cells (Tregs) as compared to untreated subjects or subjectstreated with antibodies directed to FLT3L, the Type II protein, and/ortheir respective ligands or receptors. In embodiments, the methodincreases priming of effector T cells in draining lymph nodes of thesubject as compared to untreated subjects or subjects treated withantibodies directed to FLT3L, the Type II protein, and/or theirrespective ligands or receptors. In embodiments, the method causes anoverall decrease in immunosuppressive cells and a shift toward a moreinflammatory tumor environment as compared to untreated subjects orsubjects treated with antibodies directed to the FLT3L, the Type IIprotein, and/or their respective ligands or receptors.

In embodiments, the present chimeric proteins are capable of, or can beused in methods comprising, modulating the amplitude of an immuneresponse, e.g., modulating the level of effector output. In embodiments,e.g., when used for the treatment of cancer, the present chimericproteins alter the extent of immune stimulation as compared to immuneinhibition to increase the amplitude of a T cell response, including,without limitation, stimulating increased levels of cytokine production,proliferation or target killing potential. In embodiments, the patient'sT cells are activated and/or stimulated by the chimeric protein, withthe activated T cells being capable of dividing and/or secretingcytokines.

Cancers or tumors refer to an uncontrolled growth of cells and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of the bodily organs and systems.Included are benign and malignant cancers, polyps, hyperplasia, as wellas dormant tumors or micrometastases. Also, included are cells havingabnormal proliferation that is not impeded by the immune system (e.g.,virus infected cells). The cancer may be a primary cancer or ametastatic cancer. The primary cancer may be an area of cancer cells atan originating site that becomes clinically detectable, and may be aprimary tumor. In contrast, the metastatic cancer may be the spread of adisease from one organ or part to another non-adjacent organ or part.The metastatic cancer may be caused by a cancer cell that acquires theability to penetrate and infiltrate surrounding normal tissues in alocal area, forming a new tumor, which may be a local metastasis. Thecancer may also be caused by a cancer cell that acquires the ability topenetrate the walls of lymphatic and/or blood vessels, after which thecancer cell is able to circulate through the bloodstream (thereby beinga circulating tumor cell) to other sites and tissues in the body. Thecancer may be due to a process such as lymphatic or hematogeneousspread. The cancer may also be caused by a tumor cell that comes to restat another site, re-penetrates through the vessel or walls, continues tomultiply, and eventually forms another clinically detectable tumor. Thecancer may be this new tumor, which may be a metastatic (or secondary)tumor.

The cancer may be caused by tumor cells that have metastasized, whichmay be a secondary or metastatic tumor. The cells of the tumor may belike those in the original tumor. As an example, if a breast cancer orcolon cancer metastasizes to the liver, the secondary tumor, whilepresent in the liver, is made up of abnormal breast or colon cells, notof abnormal liver cells. The tumor in the liver may thus be a metastaticbreast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originatefrom melanoma, colon, breast, or prostate, and thus may be made up ofcells that were originally skin, colon, breast, or prostate,respectively. The cancer may also be a hematological malignancy, whichmay be leukemia or lymphoma. The cancer may invade a tissue such asliver, lung, bladder, or intestinal.

Representative cancers and/or tumors of the present invention include,but are not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In embodiments, the chimeric protein is used to treat a subject that hasa treatment-refractory cancer. In embodiments, the chimeric protein isused to treat a subject that is refractory to one or moreimmune-modulating agents. For example, in embodiments, the chimericprotein is used to treat a subject that presents no response totreatment, or even progress, after 12 weeks or so of treatment. Forinstance, in embodiments, the subject is refractory to a PD-1 and/orPD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), MK-3475 (MERCK), BMS 936559 (BRISTOLMYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab(TECENTRIQ, GENENTECH), and/or MPDL328OA (ROCHE)-refractory patients.For instance, in embodiments, the subject is refractory to ananti-CTLA-4 agent, e.g., ipilimumab (YERVOY)-refractory patients (e.g.,melanoma patients). Accordingly, in embodiments the present inventionprovides methods of cancer treatment that rescue patients that arenon-responsive to various therapies, including monotherapy of one ormore immune-modulating agents.

In embodiments, the present invention provides chimeric proteins whichtarget a cell or tissue within the tumor microenviroment. Inembodiments, the cell or tissue within the tumor microenvironmentexpresses one or more targets or binding partners of the chimericprotein. The tumor microenvironment refers to the cellular milieu,including cells, secreted proteins, physiological small molecules, andblood vessels in which the tumor exists. In embodiments, the cells ortissue within the tumor microenvironment are one or more of: tumorvasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells;endothelial progenitor cells (EPC); cancer-associated fibroblasts;pericytes; other stromal cells; components of the extracellular matrix(ECM); dendritic cells; antigen presenting cells; T-cells; regulatory Tcells; macrophages; neutrophils; and other immune cells located proximalto a tumor. In embodiments, the present chimeric protein targets acancer cell. In embodiments, the cancer cell expresses one or more oftargets or binding partners of the chimeric protein.

In embodiments, the present methods provide treatment with the chimericprotein in a patient who is refractory to an additional agent, such“additional agents” being disclosed elsewhere herein, inclusive, withoutlimitation, of the various chemotherapeutic agents disclosed herein.

The activation of regulatory T cells is critically influenced bycostimulatory and co-inhibitory signals. Two major families ofcostimulatory molecules include the B7 and the tumor necrosis factor(TNF) families. These molecules bind to receptors on T cells belongingto the CD28 or TNF receptor families, respectively. Many well-definedco-inhibitors and their receptors belong to the B7 and CD28 families.

In embodiments, an immune stimulatory signal refers to a signal thatenhances an immune response. For example, in the context of oncology,such signals may enhance antitumor immunity. For instance, withoutlimitation, immune stimulatory signal may be identified by directlystimulating proliferation, cytokine production, killing activity, orphagocytic activity of leukocytes. Specific examples include directstimulation of TNF superfamily receptors such as OX40, LTbR, CD27, CD30,4-1BB or TNFRSF25 using either receptor agonist antibodies or using achimeric protein comprising the ligands for such receptors (OX40L,LIGHT, CD70, CD30L, 4-1BBL, TL1A, respectively). Stimulation from anyone of these receptors may directly stimulate the proliferation andcytokine production of individual T cell subsets. Another exampleincludes direct stimulation of an immune inhibitory cell with through areceptor that inhibits the activity of such an immune suppressor cell.This would include, for example, stimulation of CD4+FoxP3+ regulatory Tcells with a GITR agonist antibody or GITRL containing chimeric protein,which would reduce the ability of those regulatory T cells to suppressthe proliferation of conventional CD4+ or CD8+ T cells. In anotherexample, this would include stimulation of CD40 on the surface of anantigen presenting cell using a CD40 agonist antibody or a chimericprotein containing CD40L, causing activation of antigen presenting cellsincluding enhanced ability of those cells to present antigen in thecontext of appropriate native costimulatory molecules, including thosein the B7 or TNF superfamily. In another example, this would includestimulation of LTBR on the surface of a lymphoid or stromal cell using aLIGHT containing chimeric protein, causing activation of the lymphoidcell and/or production of pro-inflammatory cytokines or chemokines tofurther stimulate an immune response, optionally within a tumor.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, enhancing, restoring, promoting and/orstimulating immune modulation. In embodiments, the present chimericproteins described herein, restore, promote and/or stimulate theactivity or activation of one or more immune cells against tumor cellsincluding, but not limited to: T cells, cytotoxic T lymphocytes, Thelper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendriticcells. In embodiments, the present chimeric proteins enhance, restore,promote and/or stimulate the activity and/or activation of T cells,including, by way of a non-limiting example, activating and/orstimulating one or more T-cell intrinsic signals, including apro-survival signal; an autocrine or paracrine growth signal; a p38MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; ananti-apoptotic signal; and/or a signal promoting and/or necessary forone or more of: pro-inflammatory cytokine production or T cell migrationor T cell tumor infiltration.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, causing an increase of one or more of T cells(including without limitation cytotoxic T lymphocytes, T helper cells,natural killer T (NKT) cells), B cells, natural killer (NK) cells,natural killer T (NKT) cells, dendritic cells, monocytes, andmacrophages (e.g., one or more of M1 and M2) into a tumor or the tumormicroenvironment. In embodiments, the chimeric protein enhancesrecognition of tumor antigens by CD8+ T cells, particularly those Tcells that have infiltrated into the tumor microenvironment. Inembodiments, the present chimeric protein induces CD19 expression and/orincreases the number of CD19 positive cells (e.g., CD19 positive Bcells). In embodiments, the present chimeric protein induces IL-15Rαexpression and/or increases the number of IL-15Rα positive cells (e.g.,IL-15Ra positive dendritic cells).

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, inhibiting and/or causing a decrease inimmunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs),regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2macrophages, and tumor associated macrophages (TAMs)), and particularlywithin the tumor and/or tumor microenvironment (TME). In embodiments,the present therapies may alter the ratio of M1 versus M2 macrophages inthe tumor site and/or TME to favor M1 macrophages.

In embodiments, the present chimeric proteins are able to increase theserum levels of various cytokines or chemokines including, but notlimited to, one or more of IFNγ, TNFα, IL-2, IL-4, IL-5, IL-6, IL-7,IL-9, IL-10, IL-13, IL-15, IL-17A, IL-17F, IL-22, CCL2, CCL3, CCL4,CXCL8, CXCL9, CXCL10, CXCL11 and CXCL12. In embodiments, the presentchimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10,IL-13, IL-17A, IL-22, TNFα or IFNγ in the serum of a treated subject. Inembodiments, administration of the present chimeric protein is capableof enhancing TNFa secretion. In a specific embodiment, administration ofthe present chimeric protein is capable of enhancing superantigenmediated TNFα secretion by leukocytes. Detection of such a cytokineresponse may provide a method to determine the optimal dosing regimenfor the indicated chimeric protein.

In a chimeric protein of the present invention and/or a chimeric proteinused in methods of the present invention, the chimeric protein iscapable of increasing or preventing a decrease in a sub-population ofCD4+ and/or CD8+ T cells.

In a chimeric protein of the present invention and/or a chimeric proteinused in methods of the present invention, the chimeric protein iscapable of enhancing tumor killing activity by T cells.

In embodiments, the present chimeric proteins inhibit, block and/orreduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; orstimulate, induce, and/or increase cell death of a pro-tumor T cell. Tcell exhaustion is a state of T cell dysfunction characterized byprogressive loss of proliferative and effector functions, culminating inclonal deletion. Accordingly, a pro-tumor T cell refers to a state of Tcell dysfunction that arises during many chronic infections,inflammatory diseases, and cancer. This dysfunction is defined by poorproliferative and/or effector functions, sustained expression ofinhibitory receptors and a transcriptional state distinct from that offunctional effector or memory T cells. Exhaustion prevents optimalcontrol of infection and tumors. Illustrative pro-tumor T cells include,but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing oneor more checkpoint inhibitory receptors, Th2 cells and Th17 cells.Checkpoint inhibitory receptors refer to receptors expressed on immunecells that prevent or inhibit uncontrolled immune responses. Incontrast, an anti-tumor CD8+ and/or CD4+ T cell refers to T cells thatcan mount an immune response to a tumor.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, increasing a ratio of effector T cells toregulatory T cells. Illustrative effector T cells include ICOS+effectorT cells; cytotoxic T cells (e.g., αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺effector T cells (e.g., αβ TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi, IL⁻7R/CD127⁺); CD8⁺ effector T cells (e.g., αβ TCR, CD3⁺, CD4⁺, CCR7⁺,CD62Lhi, IL⁺7R/CD127⁺); CD8⁺ effector T cells (e.g., αβ TCR, CD3⁺, CD8⁺,CCR7⁺, CD62Lhi, IL-7R/CD127⁺); effector memory T cells (e.g., CD62Llow,CD44⁺, TCR, CD⁺, IL-7R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells(e.g., CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺,IL-7R/CD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effector memory Tcells (TEM) including early effector memory T cells (CD27⁺ CD62L⁻) andlate effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells;CD127(⁻)CD25(⁻)effector T cells; CD8⁺ stem cell memory effector cells(TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca(⁺); TH1 effectorT-cells (e.g., CXCR3⁺, CXCR6⁺ and CCR5⁺; or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺,IFNγR⁺, CXCR3⁺), TH2 effector T cells (e.g., CCR3⁺, CCR4⁺ and CCR8⁺; orαβ TCR, CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17R8⁺, CRTH2⁺); TH9effector T cells (e.g., αβ TCR, CD3⁺, CD4⁺); TH17 effector T cells(e.g., αβ TCR, CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺effector T cells, CD4⁺CD45RO⁺CCR7(⁻) effector T cells; and effector Tcells secreting IL-2, IL-4 and/or IFN-γ. Illustrative regulatory T cellsinclude ICOS⁺ regulatory T cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells,CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ regulatory T cells, CD4⁺CD25highregulatory T cells, TIM-3-PD-1⁺ regulatory T cells, lymphocyteactivation gene-3 (LAG-3)⁺ regulatory T cells, CTLA-4/CD152⁺ regulatoryT cells, neuropilin-1 (Nrp-1)⁺ regulatory T cells, CCR4⁺CCR8⁺ regulatoryT cells, CD62L (L-selectin)⁺ regulatory T cells, CD45RBlow regulatory Tcells, CD127low regulatory T cells, LRRC32/GARP⁺ regulatory T cells,CD39⁺ regulatory T cells, GITR⁺ regulatory T cells, LAP⁺ regulatory Tcells, 1B11⁺ regulatory T cells, BTLA⁺ regulatory T cells, type 1regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatorycell of natural killer T cell phenotype (NKTregs), CD8⁺ regulatory Tcells, CD8⁺CD28⁻ regulatory T cells and/or regulatory T-cells secretingIL-10, IL-35, TGF-β, TNF-α, Galectin-1, IFN-γ and/or MCP1.

In embodiments, the chimeric protein of the invention causes an increasein effector T cells (e.g., CD4+CD25− T cells).

In embodiments, the chimeric protein causes a decrease in regulatory Tcells (e.g., CD4+CD25+ T cells).

In embodiments, the chimeric protein causes an increase in CD103+antigen presenting cells (e.g., CD11c+CD103+ cells).

In embodiments, the chimeric protein generates a memory response whichmay, e.g., be capable of preventing relapse or protecting the animalfrom a recurrence and/or preventing, or reducing the likelihood of,metastasis. Thus, an animal treated with the chimeric protein is laterable to attack tumor cells and/or prevent development of tumors whenrechallenged after an initial treatment with the chimeric protein.Accordingly, a chimeric protein of the present invention and/or achimeric protein used in methods of the present invention stimulatesboth active tumor destruction and also immune recognition of tumorantigens, which are essential in programming a memory response capableof preventing relapse.

In embodiments, the chimeric protein is capable of causing activation ofantigen presenting cells. In embodiments, the chimeric protein iscapable enhancing the ability of antigen presenting cells to presentantigen.

In embodiments, the chimeric protein causes an increase in the frequencyand/or absolute numbers of CD103+ antigen presenting cells (e.g.,CD11c+CD103+ cells).

In embodiments, the chimeric protein simultaneously causes an increasein the frequency and/or absolute numbers of CD103+ antigen presentingcells (e.g., CD11c+CD103+ cells) and the activation status of those samecells (e.g., by increasing expression of CD80, and/or CD86, and/or CD40,and/or IL-12 and/or IFNg, and/or CD8).

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently stimulating effector T cells forlonger than about 12 hours, about 24 hours, about 48 hours, about 72hours or about 96 hours or about 1 week or about 2 weeks. Inembodiments, the transient stimulation of effector T cells occurssubstantially in a patient's bloodstream or in a particulartissue/location including lymphoid tissues such as for example, the bonemarrow, lymph-node, spleen, thymus, mucosa-associated lymphoid tissue(MALT), non-lymphoid tissues, or in the tumor microenvironment.

In a chimeric protein of the present invention and/or a chimeric proteinused in methods of the present invention, the present chimeric proteinunexpectedly provides binding of the extracellular domain components totheir respective binding partners with slow off rates (Kd or K_(off)).In embodiments, this provides an unexpectedly long interaction of thereceptor to ligand and vice versa. Such an effect allows for a longerpositive signal effect, e.g., increase in or activation of immunestimulatory signals. For example, the present chimeric protein, e.g.,via the long off rate binding allows sufficient signal transmission toprovide immune cell proliferation, allow for anti-tumor attack, allowssufficient signal transmission to provide release of stimulatorysignals, e.g., cytokines.

In a chimeric protein of the present invention and/or a chimeric proteinused in methods of the present invention, the chimeric protein iscapable of forming a stable synapse between cells. The stable synapse ofcells promoted by the chimeric proteins provides spatial orientation tofavor tumor reduction—such as positioning the T cells to attack tumorcells. In embodiments, this provides longer on-target (e.g.,intra-tumoral) half-life (t_(1/2)) as compared to serum t_(1/2) of thechimeric proteins. Such properties could have the combined advantage ofreducing off-target toxicities associated with systemic distribution ofthe chimeric proteins.

Additionally, the chimeric protein may independently bind to andactivate two receptors/ligands (e.g., FLT3 and a Type II transmembraneprotein's receptor/ligand) on a single immune system cell's surface.

In embodiments, the chimeric protein is capable of providing a sustainedimmunomodulatory effect.

The present chimeric proteins provide synergistic therapeutic effects(e.g., anti-tumor effects) as it allows for improved site-specificinterplay of two immunotherapy agents. In embodiments, the presentchimeric proteins provide the potential for reducing off-site and/orsystemic toxicity.

In embodiments, the present chimeric protein exhibit enhanced safetyprofiles. In embodiment, the present chimeric protein exhibit reducedtoxicity profiles. For example, administration of the present chimericproteins may result in reduced side effects such as one or more ofdiarrhea, inflammation (e.g., of the gut), or weight loss, which occurfollowing administration of antibodies directed to theligand(s)/receptor(s) targeted by the extracellular domains of thepresent chimeric proteins. In embodiments, the present chimeric proteinprovides improved safety, as compared to antibodies directed to theligand(s)/receptor(s) targeted by the extracellular domains of thepresent chimeric proteins, yet, without sacrificing efficacy.

In embodiments, the present chimeric proteins provide reducedside-effects, e.g., GI complications, relative to currentimmunotherapies, e.g., antibodies directed to ligand(s)/receptor(s)targeted by the extracellular domains of the present chimeric proteins.Illustrative GI complications include abdominal pain, appetite loss,autoimmune effects, constipation, cramping, dehydration, diarrhea,eating problems, fatigue, flatulence, fluid in the abdomen or ascites,gastrointestinal (GI) dysbiosis, GI mucositis, inflammatory boweldisease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain, stoolor urine changes, ulcerative colitis, vomiting, weight gain fromretaining fluid, and/or weakness.

In some aspects, the present chimeric agents are used to treat one ormore infections. In embodiments, the present chimeric proteins are usedin methods of treating viral infections (including, for example, HIV andHCV). In embodiments, the infections induce immunosuppression. Forexample, HIV infections often result in immunosuppression in theinfected subjects. Accordingly, as disclosed elsewhere herein, thetreatment of such infections may involve, in embodiments, modulating theimmune system with the present chimeric proteins to favor immunestimulation over blocking or preventing immune inhibition.

In embodiments, the present invention provides methods of treating viralinfections including, without limitation, acute or chronic viralinfections, for example, of the respiratory tract, of papilloma virusinfections, of herpes simplex virus (HSV) infection, of humanimmunodeficiency virus (HIV) infection, and of viral infection ofinternal organs such as infection with hepatitis viruses. Inembodiments, the viral infection is caused by a virus of familyFlaviviridae. In embodiments, the virus of family Flaviviridae isselected from Yellow Fever Virus, West Nile virus, Dengue virus,Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and HepatitisC Virus. In embodiments, the viral infection is caused by a virus offamily Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. Inembodiments, the viral infection is caused by a member ofOrthomyxoviridae, e.g., an influenza virus. In embodiments, the viralinfection is caused by a member of Retroviridae, e.g., a lentivirus. Inembodiments, the viral infection is caused by a member ofParamyxoviridae, e.g., respiratory syncytial virus, a humanparainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, andhuman metapneumovirus. In embodiments, the viral infection is caused bya member of Bunyaviridae, e.g., hantavirus. In embodiments, the viralinfection is caused by a member of Reoviridae, e.g., a rotavirus.

Combination Therapies and Conjugation

In embodiments, the invention provides for chimeric proteins and methodsthat further comprise administering an additional agent to a subject. Inembodiments, the invention pertains to co-administration and/orco-formulation. Any of the compositions disclosed herein may beco-formulated and/or co-administered.

In embodiments, any chimeric protein disclosed herein actssynergistically when co-administered with another agent and isadministered at doses that are lower than the doses commonly employedwhen such agents are used as monotherapy. In embodiments, any agentreferenced herein may be used in combination with any of the chimericproteins disclosed herein.

In embodiments, inclusive of, without limitation, cancer applications,the present invention pertains to chemotherapeutic agents as additionalagents. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and CYTOMN cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In embodiments, inclusive of, without limitation, cancer applications,the present additional agent is one or more immune-modulating agentsselected from an agent that blocks, reduces and/or inhibits PD-1 andPD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way ofnon-limiting example, one or more of nivolumab (ONO-4538/BMS-936558,MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab(TECENTRIQ, GENENTECH), MPDL328OA (ROCHE)), an agent that increasesand/or stimulates CD137 (4-1BB) and/or the binding of CD137 (4-1BB) withone or more of 4-1BB ligand (by way of non-limiting example, urelumab(BMS-663513 and anti-4-1BB antibody), and an agent that blocks, reducesand/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 withone or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and/or the bindingof OX40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK),MED16469 (MEDIMMUNE).

In embodiments, inclusive of, without limitation, infectious diseaseapplications, the present invention pertains to anti-infectives asadditional agents. In embodiments, the anti-infective is an anti-viralagent including, but not limited to, Abacavir, Acyclovir, Adefovir,Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine,Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide,Etravirine, Famciclovir, and Foscarnet. In embodiments, theanti-infective infective is an anti-bacterial agent including, but notlimited to, cephalosporin antibiotics (cephalexin, cefuroxime,cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin,cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro,Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics(tetracycline, minocycline, oxytetracycline, and doxycycline);penicillin antibiotics (amoxicillin, ampicillin, penicillin V,dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactamantibiotics (aztreonam); and carbapenem antibiotics (ertapenem,doripenem, imipenem/cilastatin, and meropenem). In embodiments, theanti-infectives include anti-malarial agents (e.g., chloroquine,quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine,atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole,tinidazole, ivermectin, pyrantel pamoate, and albendazole.

In embodiments, the chimeric proteins (and/or additional agents)disclosed herein, include derivatives that are modified, i.e., by thecovalent attachment of any type of molecule to the composition such thatcovalent attachment does not prevent the activity of the composition.For example, but not by way of limitation, derivatives includecomposition that have been modified by, inter a/ia, glycosylation,lipidation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative can contain one or more non-classical amino acids. In stillother embodiments, the chimeric proteins (and/or additional agents)disclosed herein further comprise a cytotoxic agent, comprising, inillustrative embodiments, a toxin, a chemotherapeutic agent, aradioisotope, and an agent that causes apoptosis or cell death. Suchagents may be conjugated to a composition disclosed herein.

The chimeric proteins (and/or additional agents) disclosed herein maythus be modified post-translationally to add effector moieties such aschemical linkers, detectable moieties such as for example fluorescentdyes, enzymes, substrates, bioluminescent materials, radioactivematerials, and chemiluminescent moieties, or functional moieties such asfor example streptavidin, avidin, biotin, a cytotoxin, a cytotoxicagent, and radioactive materials.

Pharmaceutical Composition

Aspects of the present invention include a pharmaceutical compositioncomprising a therapeutically effective amount of a chimeric protein asdisclosed herein.

The chimeric proteins (and/or additional agents) disclosed herein canpossess a sufficiently basic functional group, which can react with aninorganic or organic acid, or a carboxyl group, which can react with aninorganic or organic base, to form a pharmaceutically acceptable salt. Apharmaceutically-acceptable acid addition salt is formed from apharmaceutically acceptable acid, as is well known in the art. Suchsalts include the pharmaceutically acceptable salts listed in, forexample, Journal of Pharmaceutical Science, 66, 2-19 (1977) and TheHandbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, whichare hereby incorporated by reference in their entirety.

In embodiments, the compositions disclosed herein are in the form of apharmaceutically acceptable salt.

Further, any chimeric protein (and/or additional agents) disclosedherein can be administered to a subject as a component of a composition,e.g., pharmaceutical composition, that comprises a pharmaceuticallyacceptable carrier or vehicle. Such pharmaceutical compositions canoptionally comprise a suitable amount of a pharmaceutically acceptableexcipient so as to provide the form for proper administration.Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be, for example, saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea and thelike. In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be used. In embodiments, the pharmaceuticallyacceptable excipients are sterile when administered to a subject. Wateris a useful excipient when any agent disclosed herein is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid excipients, specifically forinjectable solutions. Suitable pharmaceutical excipients also includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Any agent disclosed herein, if desired, can also compriseminor amounts of wetting or emulsifying agents, or pH buffering agents.

In embodiments, the compositions, e.g., pharmaceutical compositions,disclosed herein are resuspended in a saline buffer (including, withoutlimitation TBS, PBS, and the like).

In embodiments, the chimeric proteins may by conjugated and/or fusedwith another agent to extend half-life or otherwise improvepharmacodynamic and pharmacokinetic properties. In embodiments, thechimeric proteins may be fused or conjugated with one or more of PEG,XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., humanserum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP,transferrin, and the like. In embodiments, each of the individualchimeric proteins is fused to one or more of the agents described inBioDrugs (2015) 29:215-239, the entire contents of which are herebyincorporated by reference.

The present invention includes the disclosed chimeric protein (and/oradditional agents) in various formulations of pharmaceuticalcomposition. Any chimeric protein (and/or additional agents) disclosedherein can take the form of solutions, suspensions, emulsion, drops,tablets, pills, pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. DNA or RNAconstructs encoding the protein sequences may also be used. Inembodiments, the composition is in the form of a capsule (see, e.g.,U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceuticalexcipients are described in Remington's Pharmaceutical Sciences1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated hereinby reference.

Where necessary, the pharmaceutical compositions comprising the chimericprotein (and/or additional agents) can also include a solubilizingagent. Also, the agents can be delivered with a suitable vehicle ordelivery device as known in the art. Combination therapies outlinedherein can be co-delivered in a single delivery vehicle or deliverydevice. Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection.

The pharmaceutical compositions comprising the chimeric protein (and/oradditional agents) of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodswell known in the art of pharmacy. Such methods generally include thestep of bringing therapeutic agents into association with a carrier,which constitutes one or more accessory ingredients. Typically, thepharmaceutical compositions are prepared by uniformly and intimatelybringing therapeutic agent into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product into dosage forms of the desired formulation (e.g., wet ordry granulation, powder blends, etc., followed by tableting usingconventional methods known in the art)

In embodiments, any chimeric protein (and/or additional agents)disclosed herein is formulated in accordance with routine procedures asa pharmaceutical composition adapted for a mode of administrationdisclosed herein.

Administration, Dosing, and Treatment Regimens

Routes of administration include, for example: intradermal,intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation, or topically,particularly to the ears, nose, eyes, or skin.

As examples, administration results in the release of chimeric protein(and/or additional agents) disclosed herein into the bloodstream (viaenteral or parenteral administration), or alternatively, the chimericprotein (and/or additional agents) is administered directly to the siteof active disease.

Any chimeric protein (and/or additional agents) disclosed herein can beadministered orally. Such chimeric proteins (and/or additional agents)can also be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anotherbiologically active agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment. In embodiments, for instance in thetreatment of cancer, the chimeric protein (and/or additional agents) areadministered in the tumor microenvironment (e.g., cells, molecules,extracellular matrix and/or blood vessels that surround and/or feed atumor cell, inclusive of, for example, tumor vasculature;tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelialprogenitor cells (EPC); cancer-associated fibroblasts; pericytes; otherstromal cells; components of the extracellular matrix (ECM); dendriticcells; antigen presenting cells; T-cells; regulatory T cells;macrophages; neutrophils; and other immune cells located proximal to atumor) or lymph node and/or targeted to the tumor microenvironment orlymph node. In embodiments, for instance in the treatment of cancer, thechimeric protein (and/or additional agents) are administeredintratumorally.

In embodiments, the present chimeric protein allows for a dual effectthat provides less side effects than are seen in conventionalimmunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ). For example, the present chimeric proteinsreduce or prevent commonly observed immune-related adverse events thataffect various tissues and organs including the skin, thegastrointestinal tract, the kidneys, peripheral and central nervoussystem, liver, lymph nodes, eyes, pancreas, and the endocrine system;such as hypophysitis, colitis, hepatitis, pneumonitis, rash, andrheumatic disease. Further, the present local administration, e.g.,intratumorally, obviate adverse event seen with standard systemicadministration, e.g., IV infusions, as are used with conventionalimmunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g., intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g., lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

The dosage of any chimeric protein (and/or additional agents) disclosedherein as well as the dosing schedule can depend on various parameters,including, but not limited to, the disease being treated, the subject'sgeneral health, and the administering physician's discretion. Anychimeric protein disclosed herein, can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of anadditional agent, to a subject in need thereof.

In embodiments, a chimeric protein and an additional agent(s) areadministered 1 minute apart, 10 minutes apart, 30 minutes apart, lessthan 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hoursto 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hoursapart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 daysapart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeksapart, 3 weeks apart, or 4 weeks apart.

In embodiments, the present invention relates to the co-administrationof a chimeric protein which induces an innate immune response andanother chimeric protein which induces an adaptive immune response. Insuch embodiments, the chimeric protein which induces an innate immuneresponse may be administered before, concurrently with, or subsequent toadministration of the chimeric protein which induces an adaptive immuneresponse. For example, the chimeric proteins may be administered 1minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart. In an illustrative embodiment, the chimericprotein which induces an innate immune response and the chimeric proteinwhich induces an adaptive response are administered 1 week apart, oradministered on alternate weeks (i.e., administration of the chimericprotein inducing an innate immune response is followed 1 week later withadministration of the chimeric protein which induces an adaptive immuneresponse and so forth).

The dosage of any chimeric protein (and/or additional agents) disclosedherein can depend on several factors including the severity of thecondition, whether the condition is to be treated or prevented, and theage, weight, and health of the subject to be treated. Additionally,pharmacogenomic (the effect of genotype on the pharmacokinetic,pharmacodynamic or efficacy profile of a therapeutic) information abouta particular subject may affect dosage used. Furthermore, the exactindividual dosages can be adjusted somewhat depending on a variety offactors, including the specific combination of the agents beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisease being treated, the severity of the disorder, and the anatomicallocation of the disorder. Some variations in the dosage can be expected.

For administration of any chimeric protein (and/or additional agents)disclosed herein by parenteral injection, the dosage may be about 0.1 mgto about 250 mg per day, about 1 mg to about 20 mg per day, or about 3mg to about 5 mg per day. Generally, when orally or parenterallyadministered, the dosage of any agent disclosed herein may be about 0.1mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, orabout 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg perday (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about1,100 mg, about 1,200 mg per day).

In embodiments, administration of the chimeric protein (and/oradditional agents) disclosed herein is by parenteral injection at adosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mgto about 10 mg per treatment, or about 0.5 mg to about 5 mg pertreatment, or about 200 to about 1,200 mg per treatment (e.g., about 200mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about1,200 mg per treatment).

In embodiments, a suitable dosage of the chimeric protein (and/oradditional agents) is in a range of about 0.01 mg/kg to about 100 mg/kgof body weight, or about 0.01 mg/kg to about 10 mg/kg of body weight ofthe subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg,about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kgbody weight, inclusive of all values and ranges therebetween.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al, inLiposomes in Therapy of Infectious Disease and Cancer, Lopez-Beresteinand Fidler (eds.), Liss, New York, pp. 353-365 (1989).

A chimeric protein (and/or additional agents) disclosed herein can beadministered by controlled-release or sustained-release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556; and 5,733,556, each of which is incorporated herein byreference in its entirety. Such dosage forms can be useful for providingcontrolled- or sustained-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Controlled-or sustained-release of an active ingredient can be stimulated byvarious conditions, including but not limited to, changes in pH, changesin temperature, stimulation by an appropriate wavelength of light,concentration or availability of enzymes, concentration or availabilityof water, or other physiological conditions or compounds.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al, 1985, Science 228:190; During et al, 1989, Ann.Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71:105).

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Administration of any chimeric protein (and/or additional agents)disclosed herein can, independently, be one to four times daily or oneto four times per month or one to six times per year or once every two,three, four or five years. Administration can be for the duration of oneday or one month, two months, three months, six months, one year, twoyears, three years, and may even be for the life of the subject.

The dosage regimen utilizing any chimeric protein (and/or additionalagents) disclosed herein can be selected in accordance with a variety offactors including type, species, age, weight, sex and medical conditionof the subject; the severity of the condition to be treated; the routeof administration; the renal or hepatic function of the subject; thepharmacogenomic makeup of the individual; and the specific compound ofthe invention employed. Any chimeric protein (and/or additional agents)disclosed herein can be administered in a single daily dose, or thetotal daily dosage can be administered in divided doses of two, three orfour times daily. Furthermore, any chimeric protein (and/or additionalagents) disclosed herein can be administered continuously rather thanintermittently throughout the dosage regimen.

Cells and Nucleic Acids

Aspects of the present invention provide an expression vector comprisinga nucleic acid which encodes a chimeric protein as disclosed herein. Theexpression vector comprises a nucleic acid encoding the chimeric proteindisclosed herein. In embodiments, the expression vector comprises DNA orRNA. In embodiments, the expression vector is a mammalian expressionvector.

Both prokaryotic and eukaryotic vectors can be used for expression ofthe chimeric protein. Prokaryotic vectors include constructs based on E.coli sequences (see, e.g., Makrides, Microbiol. Rev 1996, 60:512-538).Non-limiting examples of regulatory regions that can be used forexpression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 andλP_(L). Non-limiting examples of prokaryotic expression vectors mayinclude the λgt vector series such as λgt11 (Huynh et al., in “DNACloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover,ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studieret al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vectorsystems cannot perform much of the post-translational processing ofmammalian cells, however. Thus, eukaryotic host-vector systems may beparticularly useful. A variety of regulatory regions can be used forexpression of the chimeric proteins in mammalian host cells. Forexample, the SV40 early and late promoters, the cytomegalovirus (CMV)immediate early promoter, and the Rous sarcoma virus long terminalrepeat (RSV-LTR) promoter can be used. Inducible promoters that may beuseful in mammalian cells include, without limitation, promotersassociated with the metallothionein II gene, mouse mammary tumor virusglucocorticoid responsive long terminal repeats (MMTV-LTR), theβ-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).

Heat shock promoters or stress promoters also may be advantageous fordriving expression of the chimeric proteins in recombinant host cells.

In embodiments, expression vectors of the invention comprise a nucleicacid encoding the chimeric proteins, or a complement thereof, operablylinked to an expression control region, or complement thereof, that isfunctional in a mammalian cell. The expression control region is capableof driving expression of the operably linked blocking and/or stimulatingagent encoding nucleic acid such that the blocking and/or stimulatingagent is produced in a human cell transformed with the expressionvector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid. An expressioncontrol region of an expression vector of the invention is capable ofexpressing operably linked encoding nucleic acid in a human cell. Inembodiments, the cell is a tumor cell. In another embodiment, the cellis a non-tumor cell. In embodiments, the expression control regionconfers regulatable expression to an operably linked nucleic acid. Asignal (sometimes referred to as a stimulus) can increase or decreaseexpression of a nucleic acid operably linked to such an expressioncontrol region. Such expression control regions that increase expressionin response to a signal are often referred to as inducible. Suchexpression control regions that decrease expression in response to asignal are often referred to as repressible. Typically, the amount ofincrease or decrease conferred by such elements is proportional to theamount of signal present; the greater the amount of signal, the greaterthe increase or decrease in expression.

In embodiments, the present invention contemplates the use of induciblepromoters capable of effecting high level of expression transiently inresponse to a cue. For example, when in the proximity of a tumor cell, acell transformed with an expression vector for the chimeric protein(and/or additional agents) comprising such an expression controlsequence is induced to transiently produce a high level of the agent byexposing the transformed cell to an appropriate cue. Illustrativeinducible expression control regions include those comprising aninducible promoter that is stimulated with a cue such as a smallmolecule chemical compound. In other examples, the chimeric protein isexpressed by a chimeric antigen receptor containing cell or an in vitroexpanded tumor infiltrating lymphocyte, under the control of a promoterwhich is sensitive to antigen recognition by the cell, and leads tolocal secretion of the chimeric protein in response to tumor antigenrecognition. Particular examples can be found, for example, in U.S. Pat.Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which isincorporated herein by reference in its entirety.

Expression control regions and locus control regions include full-lengthpromoter sequences, such as native promoter and enhancer elements, aswell as subsequences or polynucleotide variants which retain all or partof full-length or non-variant function. As used herein, the term“functional” and grammatical variants thereof, when used in reference toa nucleic acid sequence, subsequence or fragment, means that thesequence has one or more functions of native nucleic acid sequence(e.g., non-variant or unmodified sequence).

As used herein, “operable linkage” refers to a physical juxtaposition ofthe components so described as to permit them to function in theirintended manner. In the example of an expression control element inoperable linkage with a nucleic acid, the relationship is such that thecontrol element modulates expression of the nucleic acid. Typically, anexpression control region that modulates transcription is juxtaposednear the 5′ end of the transcribed nucleic acid (i.e., “upstream”).Expression control regions can also be located at the 3′ end of thetranscribed sequence (i.e., “downstream”) or within the transcript(e.g., in an intron). Expression control elements can be located at adistance away from the transcribed sequence (e.g., 100 to 500, 500 to1000, 2000 to 5000, or more nucleotides from the nucleic acid). Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, andtypically a TATA box located 25-30 base pairs upstream of thetranscription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A promoterwill also typically contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as promoters are the promoters from mammalian viralgenes, since the viral genes are often highly expressed and have a broadhost range. Examples include the SV40 early promoter, mouse mammarytumor virus LTR promoter, adenovirus major late promoter, herpes simplexvirus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40. Introns may also be included in expressionconstructs.

There are a variety of techniques available for introducing nucleicacids into viable cells. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, polymer-based systems,DEAE-dextran, viral transduction, the calcium phosphate precipitationmethod, etc. For in vivo gene transfer, a number of techniques andreagents may also be used, including liposomes; natural polymer-baseddelivery vehicles, such as chitosan and gelatin; viral vectors are alsosuitable for in vivo transduction. In some situations, it is desirableto provide a targeting agent, such as an antibody or ligand specific fora tumor cell surface membrane protein. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al, J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al, Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990).

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al, TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Inaddition, direct and targeted genetic integration strategies may be usedto insert nucleic acid sequences encoding the chimeric fusion proteinsincluding CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editingtechnologies.

In embodiments, the expression vectors for the expression of thechimeric proteins (and/or additional agents) are viral vectors. Manyviral vectors useful for gene therapy are known (see, e.g., Lundstrom,Trends Biotechnol., 21: 1 17, 122, 2003. Illustrative viral vectorsinclude those selected from Antiviruses (LV), retroviruses (RV),adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, thoughother viral vectors may also be used. For in vivo uses, viral vectorsthat do not integrate into the host genome are suitable for use, such asa viruses and adenoviruses. Illustrative types of a viruses includeSindbis virus, Venezuelan equine encephalitis (VEE) virus, and SemlikiForest virus (SFV). For in vitro uses, viral vectors that integrate intothe host genome are suitable, such as retroviruses, AAV, andAntiviruses. In embodiments, the invention provides methods oftransducing a human cell in vivo, comprising contacting a solid tumor invivo with a viral vector of the invention.

Aspects of the present invention include a host cell comprising theexpression vector which comprises the chimeric protein disclosed herein.

Expression vectors can be introduced into host cells for producing thepresent chimeric proteins. Cells may be cultured in vitro or geneticallyengineered, for example. Useful mammalian host cells include, withoutlimitation, cells derived from humans, monkeys, and rodents (see, forexample, Kriegler in “Gene Transfer and Expression: A LaboratoryManual,” 1990, New York, Freeman & Co.). These include monkey kidneycell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); humanembryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned forgrowth in suspension culture, Graham et al, J Gen Virol 1977, 36:59);baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamsterovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells(Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g.,NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African greenmonkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g.,MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells(e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51). Illustrative cancer cell types for expressing thechimeric proteins disclosed herein include mouse fibroblast cell line,NI H3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytomacell line, P815, mouse lymphoma cell line, EL4 and its ovalbumintransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcomacell line, MC57, and human small cell lung carcinoma cell lines, SCLC #2and SCLC #7.

Host cells can be obtained from normal or affected subjects, includinghealthy humans, cancer patients, and patients with an infectiousdisease, private laboratory deposits, public culture collections such asthe American Type Culture Collection (ATCC), or from commercialsuppliers.

Cells that can be used for production of the present chimeric proteinsin vitro, ex vivo, and/or in vivo include, without limitation,epithelial cells, endothelial cells, keratinocytes, fibroblasts, musclecells, hepatocytes; blood cells such as T lymphocytes, chimeric antigenreceptor expressing T cells, tumor infiltrating lymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils,megakaryocytes, granulocytes; various stem or progenitor cells, inparticular hematopoietic stem or progenitor cells (e.g., as obtainedfrom bone marrow), umbilical cord blood, peripheral blood, and fetalliver. The choice of cell type depends on the type of tumor orinfectious disease being treated or prevented, and can be determined byone of skill in the art.

Production and purification of Fc-containing macromolecules (such asmonoclonal antibodies) has become a standardized process, with minormodifications between products. For example, many Fc containingmacromolecules are produced by human embryonic kidney (HEK) cells (orvariants thereof) or Chinese Hamster Ovary (CHO) cells (or variantsthereof) or in some cases by bacterial or synthetic methods. Followingproduction, the Fc containing macromolecules that are secreted by HEK orCHO cells are purified through binding to Protein A columns andsubsequently ‘polished’ using various methods. Generally speaking,purified Fc containing macromolecules are stored in liquid form for someperiod of time, frozen for extended periods of time or in some caseslyophilized. In embodiments, production of the chimeric proteinscontemplated herein may have unique characteristics as compared totraditional Fc containing macromolecules. In certain examples, thechimeric proteins may be purified using specific chromatography resins,or using chromatography methods that do not depend upon Protein Acapture. In embodiments, the chimeric proteins may be purified in anoligomeric state, or in multiple oligomeric states, and enriched for aspecific oligomeric state using specific methods. Without being bound bytheory, these methods could include treatment with specific buffersincluding specified salt concentrations, pH and additive compositions.In other examples, such methods could include treatments that favor oneoligomeric state over another. The chimeric proteins obtained herein maybe additionally ‘polished’ using methods that are specified in the art.In embodiments, the chimeric proteins are highly stable and able totolerate a wide range of pH exposure (between pH 3-12), are able totolerate a large number of freeze/thaw stresses (greater than 3freeze/thaw cycles) and are able to tolerate extended incubation at hightemperatures (longer than 2 weeks at 40 degrees C.). In embodiments, thechimeric proteins are shown to remain intact, without evidence ofdegradation, deamidation, etc. under such stress conditions.

Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, ornon-human primate, such as a monkey, chimpanzee, or baboon. Inembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g., GFP). In embodiments,the subject and/or animal is a transgenic animal comprising afluorescent cell.

In embodiments, the subject and/or animal is a human. In embodiments,the human is a pediatric human. In embodiments, the human is an adulthuman. In embodiments, the human is a geriatric human. In embodiments,the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore theinvention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

Kits and Medicaments

Aspects of the present invention provide kits that can simplify theadministration of any agent disclosed herein.

An illustrative kit of the invention comprises any chimeric proteinand/or pharmaceutical composition disclosed herein in unit dosage form.In embodiments, the unit dosage form is a container, such as apre-filled syringe, which can be sterile, containing any agent disclosedherein and a pharmaceutically acceptable carrier, diluent, excipient, orvehicle.

The kit can further comprise a label or printed instructions instructingthe use of any agent disclosed herein. The kit may also include a lidspeculum, topical anesthetic, and a cleaning agent for theadministration location. The kit can also further comprise one or moreadditional agent disclosed herein. In embodiments, the kit comprises acontainer containing an effective amount of a composition of theinvention and an effective amount of another composition, such thosedisclosed herein.

Aspects of the present invention include use of a chimeric protein asdisclosed herein in the manufacture of a medicament, e.g., a medicamentfor treatment of cancer and/or treatment of an inflammatory disorder dueto viral infection.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1. Construction and Characterization of an IllustrativeFLT3L- and 4-1BBL-Based Chimeric Protein

A construct encoding a murine FLT3L- and 4-1BBL-based chimeric proteinwas generated. The “mFLT3L-Fc-4-1BBL” construct included a murineextracellular domain (ECD) of FLT3L fused to a murine ECD of 4-1BBL viaa hinge-CH2-CH3 Fc domain derived from IgG1. See, FIG. 1D.

The mFLT3L-Fc-4-1BBL construct was transiently expressed in 293 cellsand purified using protein-A affinity chromatography. Western blotanalyses were performed to validate the detection and binding of allthree components of mFLT3L-Fc-4-1BBL with their respective bindingpartners (FIG. 3A). The Western blots indicated the presence of adominant multimeric band in the non-reduced lanes (FIG. 3A, lane 2 ineach blot), which was reduced to a glycosylated monomeric band in thepresence of the reducing agent, 3-mercaptoethanol (FIG. 3A, lane 3 ineach blot). As shown in FIG. 3A, lane 4 in each blot, the chimericprotein ran as a monomer at the predicted molecular weight of about 70kDa in the presence of both a reducing agent (3-mercaptoethanol) and andeglycosylation agent.

Functional ELISA (enzyme-linked immunosorbent assay) were performed todemonstrate the binding affinity of the different domains of themFLT3L-Fc-4-1BBL chimeric protein to their respective binding partners.As shown in FIG. 3B, binding of the mFLT3L domain of themFLT3L-Fc-4-1BBL chimeric protein was characterized by capturing to aplate-bound recombinant mouse mFLT3 protein and detecting via ananti-mFLT3L antibody and HRP staining. Recombinant mFLT3L protein wasused to generate a standard curve. The data shown in FIG. 3Bdemonstrates that the mFLT3L domain of the mFLT3L-Fc-4-1BBL chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity. As shown in FIG.3C, binding of the mFc portion of the mFLT3L-Fc-4-1BBL chimeric proteinwas characterized by capturing the chimeric protein to a plate-boundmouse IgG Fc gamma antibody and detecting via an HRP conjugatedanti-mouse Fc antibody. A mouse whole IgG was used to generate astandard curve. As shown in FIG. 3D, binding of the m4-1BBL domain ofthe mFLT3L-Fc-4-1BBL chimeric protein was characterized by capturing toa plate-bound recombinant mouse m4-1BB protein and detecting via ananti-m4-1BBL antibody and HRP staining. Recombinant m4-1BBL protein wasused to generate a standard curve. The data shown in FIG. 3Ddemonstrates that the m4-1BBL domain of the mFLT3L-Fc-4-1BBL chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity.

Example 2. Construction and Characterization of an Illustrative FLT3L-and CD40L-based Chimeric Protein

A construct encoding a murine FLT3L- and CD40L-based chimeric proteinwas generated. The “mFLT3L-Fc-CD40L” construct included a murineextracellular domain (ECD) of FLT3L fused to a murine ECD of CD40L via ahinge-CH2-CH3 Fc domain derived from IgG1. See, FIG. 1D.

The mFLT3L-Fc-CD40L construct was transiently expressed in 293 cells andpurified using protein-A affinity chromatography. Western blot analyseswere performed to validate the detection and binding of all threecomponents of mFLT3L-Fc-CD40L with their respective binding partners(FIG. 4A). The Western blots indicated the presence of a dominantmultimeric band in the non-reduced lanes (FIG. 4A, lane 2 in each blot),which was reduced to a glycosylated monomeric band in the presence ofthe reducing agent, β-mercaptoethanol (FIG. 4A, lane 3 in each blot). Asshown in FIG. 4A, lane 4 in each blot, the chimeric protein ran as amonomer at the predicted molecular weight of about 75 kDa in thepresence of both a reducing agent (β-mercaptoethanol) and andeglycosylation agent.

Functional ELISA were performed to demonstrate the binding affinity ofthe different domains of the mFLT3L-Fc-CD40L chimeric protein to theirrespective binding partners. As shown in FIG. 4B, binding of the mFLT3Ldomain of the mFLT3L-Fc-CD40L chimeric protein was characterized bycapturing to a plate-bound recombinant mouse mFLT3 protein and detectingvia an anti-mFLT3L antibody and HRP staining. Recombinant mFLT3L proteinwas used to generate a standard curve. The data shown in FIG. 4Bdemonstrates that the mFLT3L domain of the mFLT3L-Fc-CD40L chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity. As shown in FIG.4C, binding of the mFc portion of the mFLT3L-Fc-CD40L chimeric proteinwas characterized by capturing the chimeric protein to a plate-boundmouse IgG Fc gamma antibody and detecting via an.

HRP conjugated anti-mouse Fc antibody. A mouse whole IgG was used togenerate a standard curve. As shown in FIG. 4D, binding of the mCD40Ldomain of the mFLT3L-Fc-CD40L chimeric protein was characterized bycapturing to a plate-bound recombinant mouse mCD40 protein and detectingvia an anti-mCD40L antibody and HRP staining. Recombinant mCD40L proteinwas used to generate a standard curve. The data shown in FIG. 4Ddemonstrates that the mCD40L domain of the mFLT3L-Fc-CD40L chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity.

Example 3. Construction and Characterization of an Illustrative FLT3L-and OX40L-Based Chimeric Protein

A construct encoding a murine FLT3L- and OX40L-based chimeric proteinwas generated. The “mFLT3L-Fc-OX40L” construct included a murineextracellular domain (ECD) of FLT3L fused to a murine ECD of OX40L via ahinge-CH2-CH3 Fc domain derived from IgG1. See, FIG. 1D.

The mFLT3L-Fc-OX40L construct was transiently expressed in 293 cells andpurified using protein-A affinity chromatography. Western blot analyseswere performed to validate the detection and binding of all threecomponents of mFLT3L-Fc-OX40L with their respective binding partners(FIG. 5A). The Western blots indicated the presence of a dominantmultimeric band in the non-reduced lanes (FIG. 5A, lane 2 in each blot),which was reduced to a glycosylated monomeric band in the presence ofthe reducing agent, β-mercaptoethanol (FIG. 5A, lane 3 in each blot). Asshown in FIG. 5A, lane 4 in each blot, the chimeric protein ran as amonomer at the predicted molecular weight of about 70 kDa in thepresence of both a reducing agent (β-mercaptoethanol) and andeglycosylation agent.

Functional ELISA were performed to demonstrate the binding affinity ofthe different domains of the mFLT3L-Fc-OX40L chimeric protein to theirrespective binding partners. As shown in FIG. 5B, binding of the mFLT3Ldomain of the mFLT3L-Fc-OX40L chimeric protein was characterized bycapturing to a plate-bound recombinant mouse mFLT3 protein and detectingvia an anti-mFLT3L antibody and HRP staining. Recombinant mFLT3L proteinwas used to generate a standard curve. The data shown in FIG. 5Bdemonstrates that the mFLT3L domain of the mFLT3L-Fc-OX40L chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity. As shown in FIG.5C, binding of the mFc portion of the mFLT3L-Fc-OX40L chimeric proteinwas characterized by capturing the chimeric protein to a plate-boundmouse IgG Fc gamma antibody and detecting via an HRP conjugatedanti-mouse Fc antibody. A mouse whole IgG was used to generate astandard curve. As shown in FIG. 5D, binding of the mOX40L domain of themFLT3L-Fc-OX40L chimeric protein was characterized by capturing to aplate-bound recombinant mouse mOX40 protein and detecting via ananti-mOX40L antibody and HRP staining. Recombinant mOX40L protein wasused to generate a standard curve. The data shown in FIG. 5Ddemonstrates that the mOX40L domain of the mFLT3L-Fc-OX40L chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity.

Example 4. Construction and Characterization of an Illustrative FLT3L-and GITRL-Based Chimeric Protein

A construct encoding a murine FLT3L- and GITRL-based chimeric proteinwas generated. The “mFLT3L-Fc-GITRL” construct included a murineextracellular domain (ECD) of FLT3L fused to a murine ECD of GITRL via ahinge-CH2-CH3 Fc domain derived from IgG1. See, FIG. 1D.

The mFLT3L-Fc-GITRL construct was transiently expressed in 293 cells andpurified using protein-A affinity chromatography. Western blot analyseswere performed to validate the detection and binding of all threecomponents of mFLT3L-Fc-GITRL with their respective binding partners(FIG. 6A). The Western blots indicated the presence of a dominantmultimeric band in the non-reduced lanes (FIG. 6A, lane 2 in each blot),which was reduced to a glycosylated monomeric band in the presence ofthe reducing agent, β-mercaptoethanol (FIG. 6A, lane 3 in each blot). Asshown in FIG. 6A, lane 4 in each blot, the chimeric protein ran as amonomer at the predicted molecular weight of about 70 kDa in thepresence of both a reducing agent (β-mercaptoethanol) and andeglycosylation agent.

Functional ELISA were performed to demonstrate the binding affinity ofthe different domains of the mFLT3L-Fc-GITRL chimeric protein to theirrespective binding partners. As shown in FIG. 6B, binding of the mFLT3Ldomain of the mFLT3L-Fc-GITRL chimeric protein was characterized bycapturing to a plate-bound recombinant mouse mFLT3 protein and detectingvia an anti-mFLT3L antibody and HRP staining. Recombinant mFLT3L proteinwas used to generate a standard curve. The data shown in FIG. 6Bdemonstrates that the mFLT3L domain of the mFLT3L-Fc-GITRL chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity. As shown in FIG.6C, binding of the mFc portion of the mFLT3L-Fc-GITRL chimeric proteinwas characterized by capturing the chimeric protein to a plate-boundmouse IgG Fc gamma antibody and detecting via an HRP conjugatedanti-mouse Fc antibody. A mouse whole IgG was used to generate astandard curve. As shown in FIG. 6D, binding of the mGITRL domain of themFLT3L-Fc-GITRL chimeric protein was characterized by capturing to aplate-bound recombinant mouse mGITR protein and detecting via ananti-mGITRL antibody and HRP staining. Recombinant mGITRL protein wasused to generate a standard curve. The data shown in FIG. 6Ddemonstrates that the mGITRL domain of the mFLT3L-Fc-GITRL chimericprotein effectively interacted with its binding partner in aconcentration-dependent manner and with high affinity.

Example 5. Dual ELISA Characterization

FIG. 8 shows dual ELISA assays of mFLT3L-Fc-OX40L, mFLT3L-Fc-4-1BBL, andFLT3L-Fc-CD40L chimeric proteins. Capture was performed with ananti-FLT3L antibody, followed by detection of the co-stimulatory domainwith a his-tagged recombinant protein, and visualized using ananti-his/HRP antibody.

Example 6. Characterization of the FLT3L-Fc-CD40L Chimeric Protein

The FLT3L-Fc-CD40L chimeric protein was further characterized.

FIG. 9A shows results from the Octet system for measuring affinity ofthe FLT3L-Fc-CD40L chimeric protein with mCD40-his capture. FIG. 9Bshows results from the Octet system for measuring affinity of theFLT3L-Fc-CD40L chimeric protein with mFLT3-his capture. Octet was usedto determine on-/off-rates and binding affinities to the targetreceptors. FIG. 9C shows a summary of the data of FIG. 9A and FIG. 9B.

FIG. 9D shows results of an NFkB-mCD40 luciferase reporter assay.NFkB-mCD40 reporter cell lines were generated by stably transfectingCHO-K1 cells with both a mouse CD40-expressing vector and anNFkB-luciferase reporter vector (Promega). Cells were incubated with adose titration of commercially available mCD40L-Fc (Acro Biosystems),the mFLT3-Fc-CD40L chimeric protein, or a non-CD40L containing chimericprotein (“mXXXX-Fc-OX40L”) as a negative control. After 6 hours,luminescence was quantitated on a luminometer.

FIG. 9E shows a PathHunter U2OS cell-based assay for CD40L signaling(NFkB activity, non-canonical). PathHunter U2OS cells (DiscoverX) areresponsive to both mouse and human signaling through CD40; and areanother means to inform on NFkB activity (non-canonical). Cells wereincubated with either commercial mCD40L-Fc (Acro Biosystems) or themFLT3L-Fc-CD40L chimeric protein. After 6 hours, luciferase activity wasmeasured on a luminometer.

FIG. 9D and FIG. 9E surprisingly demonstrate that the creation of thechimeric protein is accomplished without loss of activity on the CD40Lside and, indeed, with a slight increase in activity relative to asingle-sided CD40L molecule.

FIG. 9F shows proliferation of a model cell system (mFLT3over-expressing Ba/F3 cells) in response to Flt3 signaling. The dottedline indicates the maximum proliferation of the untreated cells.Significance was determined using unpaired T-test. The IL-3 dependentproB cell line, Ba/F3, is responsive to FLT3L signaling when itover-expresses the FLT3 receptor. mFLT3 over-expressing Ba/F3 cells wereincubated with a dose titration of commercially available FLT3L-Fc (AcroBiosystems) or 4 different FLT3L-based chimeric proteins. Proliferationwas assessed using the Incucyte live-cell imaging platform; measuringconfluency over time.

FIG. 9F surprisingly demonstrates that the creation of the chimericprotein is accomplished without loss of activity on the FLT3 side and,indeed, with a significant increase in activity relative to asingle-sided FLT3 molecule.

Example 7. Characterization of the FLT3L-Fc-GITRL, FLT3L-Fc-OX40L, andFLT3L-Fc-4-1BBL Chimeric Proteins

FIG. 10A shows characterization of FLT3L-Fc-GITRL activity with anNFkB-mGITR reporter cell line generated by stably transfecting CHO-K1cells with both a mouse GITR-expressing vector and NFkB-luciferasereporter vector. NFkB-mGITR reporter cell lines were generated by stablytransfecting CHO-K1 cells with both a mouse GITR-expressing vector andPromega's NFkB-luciferase reporter vector. Cells were incubated with adose titration of commercially available mGITRL-Fc (Acro Biosystems),the mFLT3-Fc-GITRL chimeric protein, or a non-GITRL containing chimericprotein (mFLT3L-Fc-OX40L) as a negative control. After 6 hours,luminescence was quantitated on a luminometer.

FIG. 10A surprisingly demonstrates that the creation of the chimericprotein is accomplished without loss of activity on the GITRL side.

The Ba/F3 assay described above was employed to measure proliferation ofother chimeric proteins. FIG. 10B shows proliferation of a model cellssystem (mFLT3 over-expressing Ba/F3 cells) in response to Flt3 signalingvia FLT3L-Fc-GITRL. FIG. 10C shows proliferation of a model cells system(mFLT3 over-expressing Ba/F3 cells) in response to Flt3 signaling viaFLT3L-Fc-OX40L. FIG. 10D shows proliferation of a model cells system(mFLT3 over-expressing Ba/F3 cells) in response to Flt3 signaling viaFLT3L-Fc-4-1BBL. For all of FIGS. 10B-D, the dotted line indicates themaximum proliferation of the untreated cells and significance wasdetermined using one-way unpaired T-test.

FIG. 10B to FIG. 10D surprisingly demonstrate that the creation of thechimeric proteins is accomplished without loss of activity on the FLT3side and, indeed, with an increase in activity relative to asingle-sided FLT3 molecule.

Example 8. In Vivo Characterization of Chimeric Protein Signaling

Mice were given 9 or 11 consecutive injections (IP) of the murine FLT3Lchimeric proteins, diluted in 0.01% mouse serum albumin (MSA; also usedas the vehicle control). Mice were euthanized on day 10 or on 12. Serumwas collected for cytokine analysis, and spleens and mesenteric lymphnodes (MLN) were isolated. Spleen weights and total lymph node cellcounts were recorded. Cell populations of dendritic cells (CD11c-F) andactivated dendritic cells (CD11c+/CD103+ and CD11c+/MHCII+(IA/IE)) wereanalyzed by flow cytometry. Serum cytokines were assessed using aProcarta multiplex kit, which was analyzed on the Luminex platform.

FIG. 11 shows in vivo dendritic cell activation by various FLT3L-basedchimeric proteins.

FIG. 12A shows in vivo serum cytokines by various FLT3L-based chimericproteins. Mice were injected for 9 or 11 consecutive days, and thenMLN/Spleens were isolated on day 10 or 12 and analyzed by flowcytometry.

FIG. 12B shows in vivo serum cytokines by various FLT3L-based chimericproteins. Mice were injected for 9 or 11 consecutive days, and thenMLN/Spleens were isolated on day 10 or 12 and analyzed by flowcytometry.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

EQUIVALENTS

While the invention has been disclosed in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments disclosed specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A chimeric protein of a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising the extracellular domain of FMS like tyrosine kinase 3 ligand(FLT3L), (b) is a linker, and (c) is a second domain comprising theextracellular domain of CD40L wherein the linker adjoins the first andthe second domains.
 2. The chimeric protein of claim 1, wherein thefirst domain comprises the entire extracellular domain of FLT3L.
 3. Thechimeric protein of claim 1, wherein the second domain comprises theentire extracellular domain of CD40L.
 4. The chimeric protein of claim1, wherein the extracellular domain of FLT3L comprises an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 57. 5. The chimeric protein of claim 1, wherein theextracellular domain of CD40L comprises an amino acid sequence that isat least 95% identical to the amino acid sequence of SEQ ID NO:
 59. 6.The chimeric protein of claim 1, wherein the linker comprises an Fcdomain derived from IgG1 or IgG4.
 7. The chimeric protein of claim 6,wherein the linker comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NO: 3.